Progress of Tumor Growth and Metastasis After Inoculation of B16FO Melanoma Cells in Kidney of Female Nude Mice Is Inhibited by a Novel Nutrient Mixture.

BACKGROUND: Tumor metastasis is a major cause for most cancer-related deaths. Melanoma is a serious cancer that metastasizes to other areas of the body, including the lungs, liver, brain, bones, or lymph nodes. Currently used cancer therapies are ineffective with a high degree of toxicity and patient mortality. Thus, any successful treatment for melanoma must target metastasis. METHODS: We studied the effect of a novel nutrient mixture (NM) containing ascorbic acid, lysine, proline, green tea extract, quercetin, and others, on the inhibition of melanoma growth and metastasis after inoculation of B16FO melanoma cells into the left kidney of female nude mice. Female athymic mice (n = 10) 8 to 10 weeks of age, were inoculated superficially in the left kidney with 5 × 105 B16FO melanoma cells in 100 µL of media. The right kidney was left untreated. After inoculation, the mice were randomly divided into 2 groups. The control group (n = 5) was fed a regular rodent chow diet, and the test group was given the same diet supplemented with 0.5% NM. The animals in control and the test groups were sacrificed 2 weeks later. Each animal's abdominal cavity was opened, and the kidneys, lungs, liver, and spleen were excised and examined for tumor growth and metastasis. RESULTS: The kidneys in the control group weighed 25% to 30% more than those in the NM group due to colonization of B16FO melanoma cells. No metastasis to the liver or spleen was observed in either of the groups. However, severe lung metastasis was observed in the control group and mild to moderate metastasis was observed in the NM group. CONCLUSION: These results show that the NM is effective in mitigating the growth of tumors in the kidney and metastases to the lung.

Modulation of MMP-2 and -9 secretion by cytokines, inducers and inhibitors in human melanoma A-2058 cells.

Melanoma, an extremely aggressive cancer, causes the most skin cancer-related deaths, due to metastasis to other areas of the body, such as lymph nodes, lungs, liver, brain or bone. It is characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions that degrade the extracellular matrix and basement membrane, allowing cancer cells to spread to distal organs. Various cytokines, mitogens, growth factors, inducers and inhibitors control MMP activities. We investigated the roles of these in regulation of MMP-2 and -9 in human melanoma A-2058 cells. Human A-2058 cells were grown in DMEM supplemented with 15% FBS and antibiotics in 24-well tissue culture plates. At near confluence, the cells were washed with PBS and incubated in serum-free media with phorbol 12-myristate 13-acetate (PMA) at 10, 25, 50 and 100 ng/ml; TNF-α and IL-1ß at 0.1, 1, 10 and 25 ng/ml; LPS at 10, 25, 50 and 100 µg/ml; epigallocatechin gallate (EGCG) and doxycycline (Dox) at 10, 25, 50 and 100 µM without and with PMA; a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract without and with PMA at 10, 50, 100, 500 and 1,000 µg/ml; actinomycin D and cyclohexamide at 2 and 4 µM; retinoic acid and dexamethasone at 50 µM. After 24 h the media were removed and analyzed for MMP-2 and MMP-9 by zymography and densitometry. Melanoma A-2058 demonstrated strong expression of MMP-2 and slight expression of MMP-9. PMA at 100 ng/ml showed no effect on MMP-2 secretion but potently upregulated MMP-9 secretion to 400% that of control. TNF-α showed no significant overall effect on expression of MMP-2 but potent dose-dependent increased MMP-9 secretion with 200% of control at 25 ng/ml. IL-1ß showed no significant effect on MMP-2 or MMP-9 secretion by A-2058 cells, except at 25 ng/ml where MMP-2 level was reduced by ~40% and MMP-9 secretion ~50%. LPS treatment showed no significant effect on MMP-2 secretion and enhanced MMP-9 secretion up to 25 µg/ml followed by decreased level. EGCG, NM and doxycycline, without and with PMA, downregulated the expression of MMP-2 and MMP-9 in a dose-dependent manner. Actinomycin D, cyclohexamide and retinoic acid had inhibitory effects on MMP-2, while dexamethasone showed slight stimulatory effect on MMP-2 secretion. Our results showed that select cytokines, mitogens and inhibitors modulated A-2058 MMP-2 and MMP-9 expression. They suggest the clinical potential of MMP inhibitors, especially the non-toxic ones, such as the nutrient mixture and its component EGCG in management of melanoma.

Modulation of MMP-2 and MMP-9 secretion by cytokines, inducers and inhibitors in human glioblastoma T-98G cells.

Brain tumors are highly aggressive, characterized by the secretion of high levels of matrix metalloproteinase (MMP)-2 and MMP-9 that degrade the extracellular matrix and basement membrane, allowing cancer cells to spread to distal organs. Various cytokines, mitogens, growth factors, inducers and inhibitors control MMP activity. We investigated the roles of these in the regulation of MMP-2 and MMP-9 in human glioblastoma T-98G cells. Human T-98G cells were grown in DME supplemented with 15% fetal bovine serum and antibiotics in 24-well tissue culture plates. At near confluence, cells were washed with phosphate-buffered saline and incubated in serum-free media with: phorbol 12-myristate 13-acetate (PMA) at 10, 25, 50 and 100 ng/ml; tumor necrosis factor (TNF)-α and interleukin (IL)-1ß at 0.1, 1, 10 and 25 ng/ml; lipopolysaccharide (LPS) at 10, 25, 50 and 100 µg/ml; epigallocatechin gallate (EGCG) and doxycycline (Dox) at 10, 25, 50 and 100 µM without and with PMA; a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract without and with PMA at 10, 50, 100, 500 and 1,000 µg/ml; actinomycin D and cyclohexamide at 2 and 4 µM; retinoic acid and dexamethasone at 50 µM. After 24 h the media were removed and analyzed for MMP-2 and MMP-9 by zymography and densitometry. Glioblastoma T-98G cells expressed only one band corresponding to MMP-2. PMA treatment showed increased MMP-2 and MMP-9 secretions up to 25 ng/ml and decreased levels of secretions at 50 and 100 ng/ml, with no significant overall effect. TNF-α induced an up and down effect on MMP-2 and a slight induction of MMP-9. IL-1ß demonstrated a slight dose-dependent increase in T-98G secretion of MMP-2, but no induction of MMP-9. LPS showed dose-dependent decreased inactive MMP-2 secretion, increased active MMP-2 secretion and no effect on MMP-9. EGCG, Dox and NM, without and with PMA, downregulated the expression of MMP-2 and MMP-9 in a dose-dependent manner. Actinomycin D, cyclohexamide, retinoic acid and dexamethasone also had inhibitory effects on MMP-2. Our results showed that cytokines, mitogens and inhibitors modulated T-98G cell MMP-2 and MMP-9 expression, suggesting the clinical use of MMP inhibitors, particularly such potent and non-toxic ones as the nutrient mixture and its component EGCG in the management of glioblastoma cancers.

Effect of a nutrient mixture on the localization of extracellular matrix proteins in HeLa human cervical cancer xenografts in female nude mice.

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 3×106 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, the tumors 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.

Modulation of uPA, MMPs and their inhibitors by a novel nutrient mixture in human glioblastoma cell lines.

Brain tumors are highly aggressive tumors that are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions that degrade the extracellular matrix (ECM) and basement membrane, allowing cancer cells to spread to distal organs. Proteases play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of urokinase plasminogen activators (uPA) and MMPs with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs). Our main objective was to study the effect of a nutrient mixture (NM) on the activity of uPA, MMPs and TIMPs in various human gliomas. Human glioblastoma (LN-18, T-98G and A-172) cell lines (ATCC) were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1000 µg/ml. Analysis of uPA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. Glioblastoma cell lines LN-18 and T-98G expressed uPA, which was inhibited by NM in a dose-dependent manner. However, no bands corresponding to uPA were detected for the A-172 cell line. On gelatinase zymography, all three cell lines showed bands corresponding to MMP-2 and LN-18 and T-98G showed PMA (100 ng/ml)-induced MMP-9. NM inhibited their expression in a dose-dependent manner. Activity of TIMP-2 was upregulated by NM in all glioma cell lines in a dose-dependent manner. Analysis revealed a positive correlation between uPA and MMP-2 and a negative correlation between uPA/MMPs and TIMP-2. These findings suggest the therapeutic potential of NM in the treatment of gliomas.

Prevention of amiodarone-induced cardiac toxicity in male BALB/c mice by a nutrient mixture.

Amiodarone (Amio), a potent anti-arrhythmic drug, is associated with life-threatening pulmonary toxicity involving fibroses and inflammation. A unique nutrient mixture (NM) consisting of lysine, proline, ascorbic acid, N-acetyl cysteine and green tea extract has previously been shown to exhibit a broad spectrum of pharmacological, therapeutic, cardiovascular and chemopreventive properties. The present study was undertaken to determine whether the NM exhibits preventive effects on Amio-induced cardiac toxicity. Six-week-old male BALB/c mice were divided into four groups (A-D) of six animals per group. Mice in groups A and C were fed a regular diet for three weeks, while the diets of the mice in groups B and D were supplemented with 1% NM during that period. After three weeks, the mice in groups C and D received daily Amio injections of 50 mg/kg body weight intraperitoneally for 4 days, whilst those in groups A and B received saline alone. At 24 h after the final dose, mice were sacrificed, blood was withdrawn and serum was collected for clinical chemistry of the heart enzymes creatine phosphokinase (CPK) and aspartate aminotransferase (AST). In addition, livers, kidneys, hearts and lungs were excised and weighed. No significant differences in weight gain were identified among the groups and liver, kidney, heart and lung weights were comparable in all four groups. Administration of Amio to group C resulted in a significant increase in serum CPK levels, whereas in NM-fed group D, the CPK levels were comparable to those in the saline injection groups, A and B. Amio administration also resulted in a significant increase in serum AST levels in group C, but not in the group D animals which exhibited similar levels to those of groups A and B. Therefore, the results indicate that NM has the potential to protect against Amio-induced cardiac toxicity.

Modulation of u-PA, MMPs and their inhibitors by a novel nutrient mixture in human lung cancer and mesothelioma cell lines.

Lung cancer, the most prevalent cancer worldwide and malignant mesothelioma are highly aggressive tumors that are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretion. Proteases play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of u-PA and MMPs with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPS). Our main objective was to study the effect of a nutrient mixture (NM) on the activity of u-PA, MMPs and TIMPs on human lung and malignant mesothelioma (MM) cell lines. Human lung cancer (A-549 and Calu-3) and malignant mesothelioma (MSTO-211H) cell lines were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1000 µg/ml. Analysis of u-PA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. Both lung cancer cell lines expressed u-PA, which was inhibited by NM in a dose-dependent manner. However, no bands corresponding to u-PA were detected for the MSTO-211H MM cell line. On gelatinase zymography, A-549 cells showed one band corresponding to MMP-2 and induction of MMP-9 with PMA (100 ng/ml) treatment. MSTO-211H showed two bands, an intense band corresponding to MMP-2 and a faint band corresponding to MMP-9; MMP-9 was enhanced significantly with PMA treatment. NM inhibited their expression in both cell lines in a dose-dependent manner. Calu-3 showed no MMP-2 or MMP-9 expression. Activity of TIMPs was upregulated by NM in all cancer cell lines in a dose-dependent manner. Analysis revealed a positive correlation between u-PA and MMPs and a negative correlation between u-PA/MMPs and TIMPs. These findings suggest the therapeutic potential of NM in the treatment of lung and mesothelioma cancers.

Ascorbate supplementation inhibits growth and metastasis of B16FO melanoma and 4T1 breast cancer cells in vitamin C-deficient mice.

Degradation of the extracellular matrix (ECM) plays a critical role in the formation of tumors and metastasis and has been found to correlate with the aggressiveness of tumor growth and invasiveness of cancer. Ascorbic acid, which is known to be essential for the structural integrity of the intercellular matrix, is not produced by humans and must be obtained from the diet. Cancer patients have been shown to have very low reserves of ascorbic acid. Our main objective was to determine the effect of ascorbate supplementation on metastasis, tumor growth and tumor immunohistochemistry in mice unable to synthesize ascorbic acid [gulonolactone oxidase (gulo) knockout (KO)] when challenged with B16FO melanoma or 4T1 breast cancer cells. Gulo KO female mice 36-38 weeks of age were deprived of or maintained on ascorbate in food and water for 4 weeks prior to and 2 weeks post intraperitoneal (IP) injection of 5x105 B16FO murine melanoma cells or to injection of 5x105 4T1 breast cancer cells into the mammary pad of mice. Ascorbate-supplemented gulo KO mice injected with B16FO melanoma cells demonstrated significant reduction (by 71%, p=0.005) in tumor metastasis compared to gulo KO mice on the control diet. The mean tumor weight in ascorbate supplemented mice injected with 4T1 cells was reduced by 28% compared to tumor weight in scorbutic mice. Scorbutic tumors demonstrated large dark cores, associated with increased necrotic areas and breaches to the tumor surface, apoptosis and matrix metalloproteinase-9 (MMP-9), and weak, disorganized or missing collagen I tumor capsule. In contrast, the ascorbate-supplemented group tumors had smaller fainter colored cores and confined areas of necrosis/apoptosis with no breaches from the core to the outside of the tumor and a robust collagen I tumor capsule. In both studies, ascorbate supplementation of gulo KO mice resulted in profoundly decreased serum inflammatory cytokine interleukin (IL)-6 (99% decrease, p=0.01 in the B16F0 study and 85% decrease, p=0.08 in the 4T1 study) compared to the levels in gulo KO mice deprived of ascorbate. In the B16FO study, ascorbate supplementation of gulo KO mice resulted in profoundly decreased serum VEGF (98% decrease, p=0.019 than in the scorbutic gulo KO mice). As expected, mean serum ascorbate level in ascorbate-restricted mice was 2% (p<0.001) of the mean ascorbate levels in supplemented mice. In conclusion, ascorbate supplementation hinders metastasis, tumor growth and inflammatory cytokine secretion as well as enhanced encapsulation of tumors elicited by melanoma and breast cancer cell challenge in gulo KO mice.

Antineoplastic activity of a nutrient mixture in Y-79 malignant retinoblastoma cells.

Retinoblastoma is one of the most common ocular malignancies in children under the age of six. Occasionally, retinoblastoma metastasizes to extraocular organs including the bone, lung and brain. Left untreated, retinoblastoma is fatal. At present, there is no effective treatment for metastatic retinoblastoma. We investigated the antineoplastic activity of a nutrient mixture (NM) (lysine, proline, ascorbic acid and green tea extract) at concentrations of 10, 50, 100, 500 and 1,000 µg/ml in triplicate at each dose in the human malignant retinoblastoma Y-79 cell line. The parameters used were cell proliferation, expression of matrix metalloproteinases (MMPs), invasion through Matrigel, morphology and apoptosis. Cell viability was assessed by trypan blue dye exclusion test. Invasion was evaluated through Matrigel and MMP activity by gelatinase zymography. H&E staining for morphological cell alterations and apoptotic studies using the Live Green Poly Caspase Detection kit were also conducted. The nutrient mixture at 10-100 µg/ml demonstrated approximately 25% toxicity towards Y-79 retinoblastoma cells and significant toxicity at 500 and 1,000 µg/ml. The Y-79 cells secreted only MMP-2 as demonstrated by zymography; the nutrient mixture had no effect on MMP-2 expression up to 100 µg/ml, but completely blocked it at 500 µg/ml. Importantly, Y-79 retinoblastoma cells were not invasive through Matrigel. H&E staining showed cell morphological changes related to apoptosis, which was confirmed using the Live Green Poly Caspase Detection kit. Our results suggest that this nutrient mixture, which inhibited cell proliferation, expression of MMP-2 and induced apoptosis, may be a candidate for further exploration for its therapeutic potential in metastatic retinoblastoma.

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

Metastasis, commonly to the lung, is the major cause of mortality from testicular cancer. The aim of the present study was to examine the effect of a novel nutrient mixture (NM) containing ascorbic acid, amino acids and green tea extract on the inhibition of melanoma growth and metastasis using a model of intratesticular inoculation of B16FO cells into nude mice. Male athymic mice (n=12), 10-12 weeks of age, were inoculated with 5×10(5) B16FO melanoma cells in 100 µl of PBS into the right testis, while the left testis was left untreated. Following inoculation, the mice were randomly divided into two groups. The control group (n=6) was fed a regular mouse chow diet and the NM 1% group (n=6) the same diet, but supplemented with 1% NM. Four weeks later the mice were sacrificed and the abdominal cavity was opened. Mice in the control group exhibited extensive metastasis in the peritoneal cavity and severely enlarged right testes and necrotic seminiferous tubules. By contrast, in the NM 1% fed group there was no evidence of peritoneal metastasis in 50% of the animals and mild metastasis in the remaining 50%. The right testes were enlarged and seminiferous tubules in the area of invasion showed evidence of degeneration. No metastasis to the liver, kidney or spleen were evident in either group. However, severe lung metastasis was observed in 2 of 6 mice in the control group and mild metastasis in 2 of 6 mice in the NM 1% group. In conclusion, these results confirm earlier studies and verify the anti-metastatic potential of NM.

A combination of green tea extract, specific nutrient mixture and quercetin: An effective intervention treatment for the regression of N-methyl-N-nitrosourea (MNU)-induced mammary tumors in Wistar rats.

Mammary tumors were developed by intraperitoneal injection of N-methyl-N-nitrosourea (MNU) in 21-day-old, sexually immature female Wistar rats. Injection of MNU was repeated 14 weeks after the first one. When palpable tumors were evident in all of the rats, various dietary treatments were initiated for a period of 8 weeks. The treatments were designed to provide 30 mg green tea extract either alone or as a nutrient mixture (E). E was then expanded to include either a nutrient supplement (N), quercetin (Q) or both (N+Q). At the end of the treatment, tumor size/rat measured in the live rats was significantly lower in the groups receiving E, E+Q, E+N and E+N+Q than in the positive control (PC) group which did not receive any dietary treatment. Tumor number/rat, tumor volume/rat and tumor weight/rat were evaluated after sacrificing the rats on the 60th day. The rats receiving E+N+Q showed significantly lower values for the three parameters as compared to the PC group. The PC group showed 24 carcinomas mostly of grade III severity, while the E+N+Q group had only 6 carcinomas, all of which were of grade II severity.

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 < 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.

Suppression of growth and hepatic metastasis of murine B16FO melanoma cells by a novel nutrient mixture.

Highly metastatic melanoma is resistant to existing therapies. Our main objective was to investigate the effect of a nutrient mixture (NM) on B16FO tumor growth and hepatic metastasis. Tumor growth was studied in athymic nude male mice, 5-6 weeks old, inoculated with 10(6) B16FO melanoma cells subcutaneously and fed either a regular diet or one supplemented with 0.5% NM. Four weeks later, the mice were sacrificed and their tumors excised, weighed and processed for histology. Metastasis was studied in C57BL/6 mice, which received 10(6) B16FO melanoma cells by intrasplenic injection, as well as a regular or 0.5% NM-supplemented diet for 2 weeks. Survival was studied in C57BL/6 mice receiving 10(6) B16FO melanoma cells intraperitoneally (i.p.) followed by the regular, NM-supplemented, or regular diet in addition to being administered with 2 mg NM injection 3 times per week. NM inhibited the growth of B16FO melanoma cells by 50%. Lesions in the two groups were consistent with malignant melanoma. Mice were injected with B16FO cells in the spleen. Those fed the regular diet developed large black spleens and livers indicating growth in the spleen and metastasis to the liver. In contrast, mice supplemented with NM showed less growth in spleen, but also reduced metastasis to the liver. The survival time of mice receiving NM supplementation and B16FO cells i.p. was greater than in mice which were fed the regular diet. To confirm effects in vivo, we investigated the effect of NM on murine B16FO melanoma cells in vitro, including cell proliferation by MTT assay, morphology by hematoxylin and eosin (H&E) staining and apoptosis using live green caspase detection kit. In vitro, NM was not toxic at 100 microg/ml concentration, but exhibited 44% toxicity over the control at 500 and 1000 microg/ml. H&E did not indicate any changes up to 100 microg/ml. NM induced slight apoptosis at 100 microg/ml, moderate at 500 and extensive at 1000 microg/ml concentration.

Extracellular matrix-mediated control of aortic smooth muscle cell growth and migration by a combination of ascorbic acid, lysine, proline, and catechins.

Extracellular matrix (ECM) function and structure are severely compromised at atherosclerotic lesion sites, contributing to initiation and progression of the disease. This study investigated whether ECM biological properties would be beneficially affected by exposure to nutrients essential for collagen synthesis and posttranslational modification. Confluent layers of human aortic smooth muscle cells (SMC) grown on collagen substrate were cultured in the presence of the tested compounds for 7 to 10 days. Pretreated cells were removed from the ECM surface by differential treatment and replaced with secondary innocent SMC cultures. Secondary SMC growth rate and invasiveness were assayed in standard growth medium. ECM protein composition was assayed immunochemically. ECM produced in the presence of ascorbic acid reduced SMC proliferation in a dose-dependent manner. Plant-derived phenolic extracts expressed different degrees of SMC growth inhibition when present during ECM production. A combination of selected nutrients had a greater effect than did individual components. The ECM deposited by SMC in the presence of ascorbate, lysine, proline, and green tea catechins inhibited SMC migration rate up to 70%. The ECM produced under conditions of chronic essential nutrient deficiency can support proatherosclerotic SMC behavior. A combination of selected nutrients can counteract these adverse effects stronger than individual components.

Inhibitory effects of a nutrient mixture on human testicular cancer cell line NT 2/DT matrigel invasion and MMP activity.

Current treatment of testicular cancer is associated with secondary malignancy, infertility, and cytotoxicity. Based on reported antimetastatic potential, we investigated the effect of a nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid, and green tea extract on human testis cancer cell line NT 2/DT by measuring cell proliferation/cytotoxicity, modulation of MMP-2 and MMP-9 secretion, and cancer cell invasive potential. Human testis cancer cells NT 2/DT (ATCC) were grown in DME medium. At near confluence, the cells were treated with NM dissolved in media and tested at 0,10, 50, and 100 microg/mL in triplicate at each dose. Cells were also treated with PMA 200 ng/mL to study enhanced secretion of MMP-9. Cell proliferation/cytotoxicity was evaluated by MTT assay, MMP activity by gelatinase zymography, and invasion through Matrigel. The nutrient mixture showed no significant effect on testis cancer cell growth. Zymography demonstrated secretion of MMP-2 by untreated human testis cancer cells and MMP-9 with PMA induction. NM inhibited secretion of both MMPs in a dose-dependent fashion with virtual total inhibition of MMP-9 at 100 microg/mL. Invasion of human testis cancer cells through Matrigel was reduced by 84% at 50 microg/mL and at 100 microg/mL (p = 0.004). NM significantly inhibited MMP secretion and matrix invasion in testicular cancer cells without toxic effect, indicating potential as an anticancer agent.

Inhibition of glioma cell line A-172 MMP activity and cell invasion in vitro by a nutrient mixture.

Standard multimodality therapy of gliomas is associated with poor patient survival and significant toxicity. Abnormal expression of matrix metalloproteinases is associated with tumor growth and invasion. Based on reported antitumor properties, we investigated the effect of a combination of natural compounds (NM), primarily composed of lysine, proline, ascorbic acid, and green tea extract in vitro on glioma cell line A-172, by measuring MMP secretion, invasion through Matrigel, and cell proliferation. Glioma cells A-172 (ATCC) were grown in modified Dulbecco's Eagle medium with 10% fetal bovine serum and antibiotics and treated with NM at 0, 10, 50, 100, 500, and 1000 microg/mL concentration in triplicate at each dose. Cell proliferation was assayed by MTT, MMP secretion by zymography, invasion through Matrigel, and morphology by H&E staining. Zymography showed one band corresponding to MMP-2, which was inhibited by NM in a dose-dependent fashion, with virtual total inhibition at 500-microg/mL concentration. Invasion through Matrigel was completely inhibited at 1000 microg/mL NM. NM was not toxic to glioma cell line A-172 at lower concentrations and exhibited toxicity of 50% over the control at 1000 microg/mL. NM significantly inhibited MMP secretion and invasion-important parameters for cancer prevention, suggesting a possible therapeutic role.

Anti-atherogenic effects of a mixture of ascorbic acid, lysine, proline, arginine, cysteine, and green tea phenolics in human aortic smooth muscle cells.

Certain drastic behavioral modifications by arterial wall smooth muscle cells (SMC) have been considered key steps in the formation of atherosclerotic lesions: massive migration of SMC from the media to the intima layer of the vessel, dedifferentiation of SMC to proliferating phenotype, and increased secretion of inflammatory cytokines as a response to inflammatory stimuli. We investigated the anti-atherogenic effects of naturally occurring compounds (ascorbic acid, green tea extract, lysine, proline, arginine, and N-acetyl cysteine) using the model of cultured aortic SMC. Cell growth was measured by DNA synthesis, cell invasiveness was measured through Matrigel, matrix metalloproteinase-2 (MMP-2) secretion was measured by zymography, and SMC secretion of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) was measured by immunochemistry. Fetal bovine serum-stimulated SMC growth was inhibited by the nutrient mixture (NM) with 85% inhibition at 100 microg/mL. A corresponding concentration of epigallocatechin gallate (EGCG; 15 microM), the most active tea phenolic, produced a significant effect but one lower than NM. NM inhibited aortic SMC Matrigel invasion in a dose-dependent manner and significantly decreased MMP-2 expression. Stimulation of SMC with tumor necrosis factor-alpha significantly increased production and secretion of such mediators of inflammation as IL-6 and MCP-1; addition of 100 microg/mL NM inhibited secretion of MCP-1 and IL-6 by 65% and 47%, respectively. These data suggest that the NM of ascorbic acid, tea phenolics, and selected amino acids has potential in blocking the development of atherosclerotic lesions by inhibiting atherogenic responses of vascular SMC to pathologic stimuli and warrants in vivo studies.

Inhibition of pulmonary metastasis of melanoma b16fo cells in C57BL/6 mice by a nutrient mixture consisting of ascorbic Acid, lysine, proline, arginine, and green tea extract.

The authors investigated the effect of a nutrient mixture (NM) on lung metastasis by B16F0 melanoma cells in C57BL/6 female mice. Mice were divided into equal groups (1 to 6) and injected via tail vein with B16F0 cells (groups 1 to 4), B16FO cells pretreated with NM (group 5), or saline (group 6). Groups 1, 3, 4, 5, and 6 were fed the control diet and group 2 the 0.5% NM supplemented diet. Groups 3 and 4 received NM intraperitoneally (IP) and intravenously (IV), respectively. Two weeks later, pulmonary metastatic colonies were counted. Pulmonary colonization was reduced by 63% in mice supplemented with NM diet, by 86% in mice receiving NM by IP and IV injections, and completely inhibited in mice injected with melanoma cells pretreated with NM. These results show that NM is effective in inhibiting the metastasis of B16FO melanoma cells.

Anticancer effect of lysine, proline, arginine, ascorbic acid and green tea extract on human renal adenocarcinoma line 786-0.

Five-year survival is limited to 60% in renal cancer patients at diagnosis. Due to the cancer's resistance to conventional treatments and associated high morbidity, we investigated the antimetastatic effects of a specific nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid and green tea extract on human renal adenocarcinoma cell line 786-0 by measuring: cell proliferation, modulation of MMP-2 and -9 secretion, and cancer cell invasive potential. Human renal cancer cell line 786-0 (ATCC) was grown in RPMI medium in 24-well tissue culture plates. At near confluence, the cells were treated with NM, 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 secretion by gelatinase zymography, and invasion through Matrigel. Zymography demonstrated MMP-2 and MMP-9 secretion by uninduced renal cancer cells with enhanced MMP-9 induced by PMA (200 ng/ml) treatment. NM inhibited the secretion of both MMPs in a dose-dependent fashion with virtual total inhibition of MMP-2 at 500-microg/ml concentration and MMP-9 at 100 microg/ml. The invasion of renal cancer cells through Matrigel was totally inhibited (p=0.0001) by NM at 1000 microg/ml concentration. Our results support a potential role for the nutrient mixture tested in the treatment of renal cell carcinoma, by inhibition of MMP-2 and MMP-9 secretion and invasion.

Effect of ascorbic acid, lysine, proline, and green tea extract on human osteosarcoma cell line MNNG-HOS xenografts in nude mice: evaluation of tumor growth and immunohistochemistry.

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 underlie neoplastic invasion and metastasis. This prompted us to investigate the effect of a diet containing lysine, proline, arginine, ascorbic acid, and green tea extract (NM) on the growth of tumors induced by implanting human osteosarcoma MNNG in athymic nude mice and the expression of MMPs, VEGF, Ki-67 and fibronectin in these tumors, as well as the production of mucin (by PAS staining). We also investigated the effect of the supplemented diet on serum ascorbic acid, total protein content, alkaline phosphatase activity, and liver enzymes. Athymic male nude mice (n = 12) were inoculated with 3 x 10(6) osteosarcoma cells MNNG-HOS and randomly divided into group A (fed a regular diet) and group B (fed a regular diet supplemented with 0.5% NM). Four weeks later, the mice were sacrificed. Results showed that NM inhibited the growth and reduced the size of tumors in nude mice. Histological evaluation revealed increased mitotic index, MMP-9, and VEGF secretion in the control group tissues. Results demonstrate that the nutrient mixture of lysine, proline, arginine, ascorbic acid, and green tea extract tested strongly suppressed the growth of tumors without adverse effects in nude mice, suggesting potential as an anticancer agent.