In vitro and in vivo inhibition of human Fanconi anemia head and neck squamous carcinoma by a phytonutrient combination.

Head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukemia are the major causes of mortality and morbidity in Fanconi anemia (FA) patients. The objective of this study was to investigate the antineoplastic activity of PB, an antineoplastic nutrient mixture (containing quercetin, curcumin, green tea, cruciferex and resveratrol) on human FA HNSCC in vitro and in vivo. Human FA HNSCC cell line OHSU-974 (Fanconi Anemia Research Fund) was cultured in RPMI medium supplemented with 20% FBS and anti-biotics. At near confluence, cells were treated in triplicate with different concentrations of PB: 0, 10, 25, 50, 75 and 100 µg/ml. Cells were also treated with PMA to induce MMP-9 activity. Cell proliferation was detected by MTT assay, secretion of MMPs by gelatinase zymography, invasion through Matrigel, migration by scratch test and morphology by hematoxylin and eosin (H&E) staining. In vivo, athymic male nude mice (n=12) were inoculated with 3x106 OHSU-974 cells subcutaneously and randomly divided into two groups: group A was fed a regular diet and group B a regular diet supplemented with 1% PB. Four weeks later, the mice were sacrificed and their tumors were excised, weighed and processed for histology. NM inhibited the growth of OHSU-974 tumor by 67.6% (p<0.0001) and tumor burden by 63.6% (p<0.0001). PB demonstrated dose-dependent inhibition of cell proliferation, with 27% (p=0.0003) and 48% (p=0.0004) toxicity at 75 and 100 µg/ml, respectively. Zymography revealed MMP-2 and PMA-induced MMP-9 secretion. PB suppressed secretion of both MMPs in a dose-dependent manner, with total block of both at 50 µg/ml. PB inhibited cell migration (by scratch test) and OHSU-974 invasion through Matrigel in a dose-dependent fashion with total block at 50 µg/ml. H&E staining showed no morphological changes below 50 µg/ml. The results suggest that PB has potential therapeutic use in the treatment of human FA HNSCC.

Effects of a nutrient mixture on immunohistochemical localization of cancer markers in human cervical cancer HeLa cell tumor xenografts in female nude mice.

Although fully treatable in the early stages, once cervical cancer has metastasized, patient outcome is poor. The main objective of this study was to examine the effect of dietary supplementation with a nutrient mixture (NM) containing lysine, ascorbic acid, proline, green tea extract and other micronutrients on HeLa cell xenografts in nude female mice. Tumor growth was measured and immunohistochemical staining was evaluated for the following cancer markers: Ki67 (proliferation); matrix metalloproteinase (MMP)-2 and -9 (invasion/metastasis); vascular endothelial growth factor (VEGF) (angiogenesis); terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and B-cell lymphoma 2 (Bcl-2) (apoptosis); cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) (inflammation); and glutathione S-transferase π (GSTπ) (a general cancer marker). Following housing for a week, 5/6-week-old female athymic nude mice (n=12) were inoculated subcutaneously with 3×106 HeLa cells in 0.2 ml phosphate-buffered saline and 0.1 ml Matrigel™ and randomly divided into two groups; control group mice were fed regular mouse chow and NM group mice the regular diet supplemented with 0.5% NM (w/w). After four weeks, the mice were sacrificed and their tumors were excised and processed for histology. The NM strongly inhibited the growth of HeLa xenografts in nude mice. The mean tumor weight was reduced to 59% (P=0.001) in the mice fed the NM compared with the tumor weight in the controlled diet mice. Ki67, MMP-2 and -9, VEGF, TUNEL, Bcl-2, COX-2, iNOS and GSTπ all showed a lower intensity and frequency of staining in the NM group compared with that in the control group. In conclusion, NM supplementation strongly inhibited tumor growth and cancer markers in female nude mice injected with HeLa xenografts.

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.

Inhibition of growth and expression of inflammation mediators in human leukemic cell line U-937 by a nutrient mixture.

AIM: A nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract has exhibited anticancer activity in vitro and in vivo in a number of cancer cell lines. We investigated the effect of NM on human leukemic myeloid U-937 cells in vitro by measuring: cell proliferation, MMP expression, invasion, apoptosis, and COX-2 and COX-1 protein expression. METHODS: Human leukemic cell line U-937 (ATCC) was cultured in RPMI medium supplemented with fetal bovine serum and antibiotics. After 24 h, the cells were treated with NM at 0, 50, 100, 250, 500 and 1000 Ојg/ml, in triplicate at each dose. Phorbol 12-myristate 13-acetate (PMA), 100 ng/ml was added to cells to induce MMP-9 secretion. Cell proliferation was evaluated by MTT assay, MMP expression by gelatinase zymography, invasion through Matrigel, apoptosis by using live green caspase detection kit (Molecular Probe), and COX-2 and COX-1 expression by Western blot. RESULTS: NM had no effect on U-937 cell growth at a concentration of 250 Ојg/ml and exhibited an antiproliferative effect at 500 Ојg/ml concentration. Zymography did not demonstrate MMP-2 or MMP-9 secretion in normal cells; however, PMA strongly induced MMP-9, which was inhibited by NM in a dose-dependent manner. Cell penetration through Matrigel was significantly reduced (by 95%) at 250 Ојg/ml NM and completely blocked at 500 Ојg/ml NM. NM induced slight apoptosis at 100 Ојg/ml and moderate at 500 and 1000 Ојg/ml concentration. NM inhibited COX-2 expression in a dose-dependent fashion and had no effect on COX-1 expression. CONCLUSIONS: Our results suggest that NM has potent inhibitory effects on U-937 cell growth and expression of inflammatory mediators, significant parameters in AML progression.

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.

In vitro and in vivo inhibition of human Fanconi anemia-associated head and neck squamous cell carcinoma by a novel nutrient mixture.

Head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukemia are the major causes of mortality and morbidity in Fanconi anemia (FA) patients. The objective of this study was to investigate the antineoplastic activity of a novel antineoplastic nutrient mixture (NM) (containing lysine, proline, ascorbic acid and green tea extract) in human FA-associated HNSCC (FA HNSCC) in vitro and in vivo. The human FA HNSCC cell line, OHSU-974 (Fanconi Anemia Research Fund), was cultured in RPMI medium supplemented with 20% FBS and antibiotics. At near confluence, cells were treated in triplicate with various concentrations of NM: 0, 50, 100, 250, 500 and 1,000 µg/ml. Cells were also treated with phorbol 12-myristate 13-acetate (PMA) to induce matrix metalloproteinase (MMP)-9 activity. Cell proliferation was detected by MTT assay, the secretion of MMPs by gelatinase zymo-graphy, cell invasion through Matrigel, cell migration by a scratch test and morphology by hematoxylin and eosin (H&E) staining. In vivo, athymic male nude mice (n=12) were inoculated with 3x106 OHSU­974 cells subcutaneously and randomly divided into 2 groups: group A was fed a regular diet and group B a regular diet supplemented with 1% NM. Four weeks later, the mice were sacrificed and their tumors were excised, weighed and processed for histological analysis. NM inhibited the growth of OHSU-974 tumors by 47% and tumor burden by 50%. At lower concentrations, NM demonstrated no effect on proliferation, but at 1,000 µg/ml a 40% toxicity was observed. Zymography revealed the MMP-2 and PMA-induced MMP-9 secretion. NM suppressed the secretion of both MMPs in a dose-dependent manner, with a virtual inhibition at 500 µg/ml. NM inhibited OHSU-974 cell invasion through Matrigel in a dose-dependent manner with a complete block at 1,000 µg/ml. H&E staining showed no morphological changes below 500 µg/ml. These results suggest that NM has potential therapeutic use in the treatment of human FA HNSCC.

Anticancer effects of a specific mixture of nutrients in the multidrug-resistant human uterine sarcoma MES-SA/Dx5 and the drug-sensitive MES-SA cell lines.

A specific nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract has demonstrated a broad spectrum of antitumor activity against a number of cancer cell lines. In this study, our main objective was to investigate the comparative effects of NM on anticancer parameters, such as cytotoxicity, matrix metalloproteinase (MMP) secretion and Matrigel invasion in the human uterine sarcoma drug-resistant MES-SA/Dx5 and the drug-sensitive MES-SA cell lines. In addition we studied the effects of NM on P-glycoprotein (Pgp) on these cell lines. Cell proliferation was evaluated by MTT assay, MMPs by gelatinase zymography, invasion through Matrigel, morphology by H&E and Pgp expression by Western blot analysis and immunodetection using FITC-conjugated antibody and rhodamine 123 (Rh123) accumulation and efflux assays. NM exhibited antiproliferative effects on MES-SA/Dx5, by 20% at 50 and 100 µg/ml and by 36, 40 and 48% at 250, 500 and 1,000 µg/ml, respectively. By contrast, NM treatment of MES-SA cells resulted in significantly increased cytotoxicity: 40, 46, 65 and 72% at 50, 100, 500 and 1,000 µg/ml, respectively. In both cell lines, zymography demonstrated a band corresponding to MMP-2 in normal cells and MMP-9 with phorbol 12-myristate 13-acetate treatment. The two MMPs showed dose-response inhibition by NM. As shown by Western blot analysis and immunodetection, NM treatment resulted in a dose-dependent decrease in Pgp expression in the MES-SA/Dx5 cell line. The MES-SA cell line does not exhibit Pgp. NM enhanced the accumulation and efflux of the Pgp substrate, Rh123, in the MES-SA/Dx5 uterine sarcoma cell line but not in the drug-sensitive cell line, MES-SA. Therefore, it can be concluded that NM demonstrates potent anticancer effects in both the drug-resistant and sensitive cell lines and modulates Pgp, suggesting its potential therapeutic effects in drug-resistant as well as sensitive cancers.

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.

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 effect of a nutrient mixture on human hepatocarcinoma cell line SK-HEP-1.

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.

Antineoplastic effects of nutrient mixture on raji and jurkat t cells: the two highly aggressive non Hodgkin's lymphoma cell lines.

UNLABELLED: 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.

Marked inhibition of growth and invasive parameters of head and neck squamous carcinoma FaDu by a nutrient mixture.

Head and neck squamous cell carcinomas (HNSCCs) are known for their aggressive growth and propensity to metastasize. The authors investigated the effect of a novel nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea extract on human HNSCC cell line FaDu in vivo and in vitro. Athymic male nude mice (n = 12) were inoculated with 3 x 10(6) FaDu cells subcutaneously and randomly divided into 2 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, weighted, and processed for histology. In vitro, FaDu cells were cultured in Dulbecco's modified Eagle's medium and exposed to NM at 0 to 1000 microg/mL in triplicate. Cell proliferation was assessed by MTT assay, matrix metalloproteinase (MMP) secretion by gelatinase zymography, invasion through Matrigel, apoptosis by live-green caspases, and cell morphology by hematoxylin-eosin staining. NM inhibited the growth of tumors by 55% (P = .0002) and exhibited dose-dependent toxicity on FaDu cells in vitro, with 53% (P = .0003) at 1000 microg/mL NM. Zymography revealed MMP-2 and phorbol 12-myristate 13-acetate-induced MMP-9 secretion. NM inhibited secretion of both MMPs in a dose-dependent manner, with virtual total inhibition at 1000 microg/mL. NM significantly inhibited FaDu invasion through Matrigel with total block at 1000 microg/mL. NM induced dose-dependent apoptosis. In conclusion, NM has therapeutic potential in the treatment of HNSCC by significantly suppressing tumor growth and significantly inhibiting MMP secretion and invasion of HNSCC cells in vitro.

A nutrient mixture suppresses carbon tetrachloride-induced acute hepatic toxicity in ICR mice.

We examined the effect of a nutrient mixture (NM) that contains lysine, proline, ascorbic acid, and green tea extract in mice treated with carbon tetrachloride (CCl4), a model of liver injury in which free radical, oxidative stress, and cytokine production are closely linked. Seven-week-old male Imprinting Control Region (ICR) mice were divided into four groups (A-D) of five animals each. Groups A and C mice were fed a regular diet for 2 weeks, whereas groups B and D mice were supplemented with 0.5% NM (w/w) during that period. Groups A and B received corn oil i.p., whereas groups C and D received CCl4 (25 microL/kg, in corn oil, i.p.). All animals were killed 24 h after CCl4 administration, serum was collected to assess liver and kidney functions, and livers and kidneys were excised for histology. Mean serum aspartate aminotransferase and alanine aminotransferase were comparable in groups A and B, increased markedly in group C, and significantly lowered in group D compared with group C. CCl4 had no significant effect on renal markers (blood urea nitrogen [BUN], creatinine, and BUN/creatinine ratio). CCl4 administration caused an intense degree of liver necrosis that was less severe in the NM fed group D. These results indicate that NM could be a useful supplement in preventing acute chemical-induced liver toxicity.

Inhibition of 7,12-dimethylbenzanthracene-induced skin tumors by a nutrient mixture.

The annual incidence of all forms of skin cancer, the most common of all human cancers, is increasing yearly. A unique nutrient mixture (NM) was shown to exhibit anticancer activity in vivo and in vitro. We examined the effect of NM on the development of skin cancer induced by 7,12-dimethylbezanthracene (DMBA) in female SENCAR mice by a complete carcinogenesis protocol. Mice (n=55) were divided into four groups and carefully shaved on dorsum. After 2 days, the mice in Groups 1 (n=10), 3 (n=20), and 4 (n=20) were treated topically with 100 nM DMBA in 0.2 ml of acetone twice a week for 4 weeks; Group 2 (n=5), the control group, was treated with acetone 0.2 ml. Groups 1 and 2 were fed the regular diet. Group 3A (n=10) was fed a diet containing 0.5% NM from the day of DMBA treatment and 3B (n=10) the regular diet and received NM (75 mg in 0.4 ml of 1:1 acetone/water) topically to the shaved area 15 min before DMBA application twice a week for 4 weeks. Group 4 mice were fed a diet containing 0.5% NM for 2 weeks prior to the application of DMBA and then divided into two groups: 4A (n=10) was fed the 0.5% NM diet as in 3A, and 4B (n=10) the regular diet as described for 3B. Body weight and diet consumption of the mice were monitored and the skin tumors (papillomas) were counted and recorded. Ten weeks thereafter the mice were euthanized, skinned, and tumors were processed for histology. NM significantly (P<0.0001) inhibited DMBA-induced skin tumor multiplicity by 59, 62, 69, and 86% in NM-treated Groups 3A, 3B, 4A, and 4B, respectively. These results suggest that NM has strong potential as a useful therapeutic regimen for skin cancer by significantly inhibiting the incidence and tumor multiplicity of DMBA-induced skin tumors.

A nutrient mixture suppresses hepatic metastasis in athymic nude mice injected with murine B16FO melanoma cells.

Highly metastatic melanoma is resistant to existing therapies. A unique micronutrient mixture (NM) containing ascorbic acid, amino acids, green tea extract has been shown to exhibit anticancer activity in vivo and in vitro in a number of cancer cell lines including human and murine melanoma cells lines. In this study we examined the effect of dietary NM supplementation on hepatic metastasis of intrasplenic injection of B16FO melanoma cells in athymic nude mice. Athymic nude mice (n = 10), 10-12 weeks of age, received 10(6) B16FO melanoma cells by injection into the spleen and divided into two groups. The Control group of mice received Purina mouse chow and the NM group received the regular diet supplemented with NM 0.5%. After two weeks, animals were sacrificed and spleens, livers, kidneys and lungs were excised from all animals, examined, weighed and processed for histology. The Control mice developed large black spleens and livers indicating growth in the spleen and metastasis to the liver. However, the mice supplemented with NM not only showed less tumor growth in the spleen as the Control mice, but also drastically reduced metastasis to the liver. In all groups, no metastasis to the kidneys and lungs was evident. In conclusion, these results suggest that NM has potential in suppression of tumor metastasis.

Vibrio cholerae metalloproteinase degrades intestinal mucin and facilitates enterotoxin-induced secretion from rat intestine.

Mucin secretion in situ from rat intestinal loops was promoted more effectively by dialysed crude cholera filtrate than by an equivalent amount of purified enterotoxin. The filtrate could be rendered inactive by incubation with mixed gangliosides or passage through a GM1-affinity column, which indicated that the secretory action of the filtrate depended upon its enterotoxin component. In an effort to explain the greater potency of the filtrate, we established the presence of a metalloproteinase in the filtrate and demonstrated that this enzyme was capable of degrading purified rat intestinal mucin. Sufficient degradation occurred to cause a substantial decrease in viscosity (57% in 120 min). Biochemical analysis of the mucin before and after exposure to filtrate revealed a rise in the combined percentage of serine, threonine and proline (53-58%), suggesting that poorly glycosylated areas (which are less abundant in these amino acids) were being partly removed from the mucin. The carbohydrate composition was essentially unaltered. Inhibition of the filtrate metalloproteinase by Zincov and alpha 2-macroglobulin significantly (P less than 0.005) reduced the ability of cholera filtrate to degrade mucin or to stimulate mucin secretion from rat intestinal slices in vitro. Purified cholera enterotoxin added to enterotoxin-depleted filtrate was a more potent secretagogue (secretory stimulant) in intestinal loops than an equivalent amount of enterotoxin alone. We therefore propose that mucin secretion induced by cholera filtrate is caused by cholera enterotoxin, but that degradation of the protective epithelial mucus layer by a constituent metalloproteinase may assist the toxin by allowing increased access to mucosal GM1 receptor sites.

Epithelial response to intestinal anaphylaxis in rats: goblet cell secretion and enterocyte damage.

The effects of immunoglobulin E (IgE)-mediated reactions on the intestinal epithelium were examined during intestinal anaphylaxis in the rat. Rats sensitized by intraperitoneal injection of egg albumin (EA) plus alum developed high serum titers of IgE anti-EA antibodies after 14 days; sham-treated littermate controls had no anti-EA antibodies. Two isolated loops of jejunum were prepared in vivo in anesthetized rats. The loops were injected with EA in saline or saline alone, and intraluminal contents of each loop were examined after 4 h. Mucosal histamine decreased in sensitized rat intestine exposed to EA. Luminal mucin, measured by radioimmunoassay, was not increased by antigen challenge. In contrast, DNA, protein, and sucrase activities were elevated in contents from the isolated segments exposed to EA in sensitized rats. Histology revealed that periodic acid-Schiff-stained material was contained in goblet cells in sections prepared from these segments after antigen exposure. Cellular debris was present over the tips of the villi. These findings suggest that IgE-mediated reactions in the intestine cause epithelial damage and loss of material from cells other than goblet cells. The results indicate that release of goblet cell mucus is not a feature of intestinal anaphylaxis.

Cholera toxin stimulates secretion of immunoreactive intestinal mucin.

In vitro secretion of goblet cell mucin from rat small intestine was measured using a double-antibody radioimmunoassay for mucin. Cholera toxin (12.5-50 mg crude filtrate/ml) added to incubations of intestinal slices caused a dose-dependent increase in mucin secretion. By 90 min there was a four- to fivefold enhancement in secretion over noncholera-treated controls. Crude filtrate (dialyzed or nondialyzed) was a more effective mucin secretogogue than purified enterotoxin. Secretion was also assessed by administering [1-14C]glucosamine intraperitoneally to rats in vivo and 3 h later monitoring in vitro secretion of radioactive glycoprotein from intestinal slices. Cholera filtrate (12.5-50 mg/ml) caused a 1.5- to 2.0-fold enhancement in secretion after 90 min. The radioactivity data, however, underestimated total mucin secretion and the dependency of secretion on the dose of cholera filtrate. Cholera preparations also caused an enhancement (20-30% over controls) in the incorporation of [3H]glucosamine into tissue acid-precipitable glycoprotein, indicating a stimulation of glycoprotein synthesis. In the same experiments it was noted that the secretion of 3H-labeled (i.e., newly glycosylated) glycoprotein was increased 2.5- to 3.0-fold over untreated controls. Assuming that radioactivity partially reflects mucin synthetic and secretory events, it is possible, therefore, that cholera toxin promotes the release of both "old" mucin from storage granules as well as the synthesis and secretion of "new" mucin formed in goblet cells during incubation.