A nutrient mixture reduced tumor growth of SK-UT-1 human leiomyosarcoma cells in vivo and in vitro by inhibiting MMPs and inducing apoptosis.

BACKGROUND: Uterine leiomyosarcoma is a rare malignant smooth muscle tumor originating in the uterine wall that generally responds poorly to chemotherapy and radiation. AIM: We investigated the in vitro effects of a novel nutrient mixture containing lysine, proline, ascorbic acid, and green tea extract on the human leiomyosarcoma cell line SK-UT-1 by measuring cell proliferation, invasiveness, apoptosis, and expression of matrix metalloproteinases (MMP). We also tested the effects of nutrient mixture in vivo using nude mice. MATERIALS AND METHODS: Human leiomyosarcoma SK-UT-1 cells were treated with different concentrations of nutrient mixture. Cell proliferation was determined by MTT assay; MMP expression by gelatinase zymography; invasion by Matrigel assay; migration by scratch test; apoptosis using Live Green caspase kit. In vivo studies were conducted on 5-6 weeks old female nude mice inoculated subcutaneously with 3 â€¢ 106 SK-UT-1 cells. The mice were fed a regular diet or a diet supplemented with 0.5% nutrient mixture. After four weeks, the mice were sacrificed and the tumors were weighed and processed for histology. RESULTS: In vitro, nutrient mixture treatment was not toxic to SK-UT-1 cells at 250 µg/ml but exhibited 20% and 40% cytotoxicity at 500 and 1000 µg/ml respectively. Zymography did not show bands for either MMP-2 or MMP-9 in SK-UT-1 cells. However, treatment with phorbol myristate acetate stimulated the expression of MMP-9, both active and inactive forms in equal proportion. Nutrient mixture inhibited the secretion of both active and inactive forms in a dose dependent manner. Invasion through Matrigel and migration by scratch test were inhibited in a dose dependent fashion, with both invasion and migration inhibited at 250 µg/ml. Live Green Caspase apoptosis assay demonstrated slight apoptosis at 100 µg/ml and significant apoptosis at 250 to 1000 µg/ml. The results of in vitro studies were further confirmed in vivo by showing 50% decrease in tumor weight, 40% reduction in tumor burden compared to the tumors from mice fed regular diet. CONCLUSION: The results suggest a therapeutic potential for nutrient mixture in uterine leiomyosarcoma treatment.

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.

Cytokines, inducers and inhibitors modulate MMP-2 and MMP­9 secretion by human Fanconi anemia immortalized fibroblasts.

Acute myeloid leukemia and head and neck squamous cell carcinomas are the major causes of mortality and morbidity in Fanconi anemia (FA) patients. Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, have been implicated in tumor invasion and metastasis. Various cytokines, mitogens, growth factors, inducers and inhibitors control MMP activities. We investigated the roles of these in the regulation of MMP-2 and MMP-9 in human immortalized fibroblasts from FA. Human FA immortalized fibroblast cell lines FA-A:PD220 and FA-D2:PD20 were grown in minimum essential medium (MEM) supplemented with 15% fetal bovine serum (FBS) and antibiotics in 24-well tissue culture plates. At near confluence, the cells were washed with phosphate­buffered saline (PBS) and incubated in serum-free media with the following: phorbol 12-myristate 13-acetate (PMA) at 10-100 ng/ml; tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) at 0.1-25 ng/ml; lipopolysaccharide (LPS) at 10-100 µg/ml; epigallocatechin gallate (EGCG) and doxycycline (Dox) at 10-100 µM without and with PMA; a nutrient mixture (NM) without and with PMA at 10-1,000 µg/ml; actinomycin-D and cyclohexamide at 2 and 4 µM; retinoic acid and dexamethasone at 50 µM. After 24 h, media were removed and analyzed for MMP-2 and MMP-9 by zymography. Both FA cell lines expressed only MMP-2 and responded similarly to cytokines, mitogens, inducers and inhibitors. PMA potently stimulated MMP-9 and had a moderate effect on MMP-2. TNF-α showed variable effects on MMP-2 and significantly enhanced MMP-9. IL-1ß enhanced MMP-2 slightly and MMP-9 significantly. LPS had a moderate stimulatory effect on MMP-2 and no effect on MMP-9. EGCG, Dox and NM, without and with PMA, downregulated MMP-2 and MMP-9 expression. Actinomycin-D, retinoic acid and dexamethasone also had inhibitory effects on MMP-2. Our results showed that cytokines, mitogens and inhibitors modulated FA fibroblast MMP-2 and MMP-9 expression, suggesting the clinical use of MMP inhibitors, particularly such potent and non-toxic ones as the NM and its component EGCG in the management of FA cancers.

A nutrient mixture inhibits glioblastoma xenograft U-87 MG growth in male nude mice.

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.

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.

In vitro and in vivo effects of a nutrient mixture on breast cancer progression.

Long-term survival of patients with breast cancer remains poor, due to metastasis and recurrence. We investigated the effects of a novel nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract in vitro and in vivo on 4T1 murine breast cancer, a representative model for metastatic breast cancer. After one week of isolation, 5-6-week-old female Balb/C mice were inoculated with 5x105 4T1 cells into the mammary pad and randomly divided into two groups; the control group was fed a regular diet and the NM group a regular diet supplemented with 0.5% NM. After four weeks, the mice were sacrificed and their tumors, lungs, livers, kidneys, hearts and spleens were excised and processed for histology. Dimensions (length and width) of tumors were measured using a digital caliper, and the tumor burden was calculated using the following formula: 0.5 x length x width. We also tested the effect of NM in vitro on 4T1 cells, measuring cell proliferation by MTT assay, MMP secretion by zymography, invasion through Matrigel, migration by scratch test and morphology by H&E staining. NM inhibited tumor weight and burden of 4T1 tumors by 50% (p=0.02) and 53.4% (p≤0.0001), respectively. Lung metastasis was profoundly inhibited by NM supplementation: mean number of colonies was reduced by 87% (p<0.0001) and mean weight of lungs by 60% (p=0.0001) compared to control mice. Metastasis to liver, spleen, kidney and heart was significantly reduced with NM supplementation. In vitro, NM exhibited 50% toxicity over the control at 250 and 500 µg/ml concentrations. Zymography demonstrated MMP-2 and MMP-9 secretion which was inhibited by NM in a dose-dependent manner, with virtual total inhibition of both at 1,000 µg/ml. Migration by scratch test and invasion through Matrigel were inhibited in a dose-dependent manner with total block of invasion at 250 and of migration at 1,000 µg/ml. These results suggest that NM has therapeutic potential in the treatment of breast cancer.

Effect of a nutrient mixture on matrix metalloproteinase-9 dimers in various human cancer cell lines.

Strong clinical and experimental evidence demonstrates association of elevated levels of matrix metalloproteinase MMP-9 with cancer progression, metastasis and shortened patient survival, as it plays a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. MMP-9 is secreted in both the monomeric and dimeric form. Although there is little research on MMP-9 dimers, some studies have shown the dimer to be associated with more aggressive tumor progression. Our objective was to study the relative secretion patterns of MMP-9 monomer and dimer in a variety of cancer cell lines and the effect of a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract on MMP-9 secretion. The cancer cell lines were grown in their respective media, supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) in 24-well tissue culture plates. At near confluence, the cells were treated with NM at 0,10, 50, 100, 500 and 1000 µg/ml. Parallel sets of cultures were treated with PMA (100 ng/ml) for induction of MMP-9. Cell MMP-9 secretion was assayed by gelatinase zymography. MMP-9 dimer secretion patterns of cancer cells fell into different categories. We observed no MMP-9 dimer in prostate DU-145 and PC-3, pancreatic MIA-Pa-Ca2, colon HCT-116, bladder T-24, head and neck FaDu, glioblastoma A-172, T-98 and LN-18 and leukemia HL-60, Jurkat, and Raji cell lines. MMP-dimer secretion only with PMA induction was seen in breast MCF-7 and MDA-MB-231, uterine SK-UT-1, lung A-549, tongue SC-25, melanoma A2058, osteosarcoma U-2OS, rhabdomyosarcoma, fibrosarcoma HT-1080, chondrosarcoma SW-1350 and liposarcoma SW-872. Cervical HeLa and DoTc 2 4510, renal 786-0 and HCC SK-Hep-1 cells exhibited MMP-9 dimer without PMA treatment and increased secretion with PMA treatment. Sarcomas had the highest levels of MMP-9 monomer and dimer with and without PMA among these cancer cell lines. Cervical, uterine and male breast cancer cell lines showed the next highest levels of MMP-9, followed by breast cancer cell lines. Melanoma, renal, lung, head and neck and HCC showed lower levels and prostate, glioblastoma, bladder and leukemia cell lines the lowest. NM showed dose-dependent inhibition of MMP-9 monomer and dimer in all cell lines tested. In conclusion, high MMP-9 and dimer secretion levels correlated with the most aggressive cancer cell lines. NM was effective in inhibiting MMP-9 and dimer secretion in all cell lines tested, suggesting its therapeutic potential as an antimetastatic agent.

In vitro modulation of MMP-2 and MMP-9 in pediatric human sarcoma cell lines by cytokines, inducers and inhibitors.

The highly aggressive pediatric sarcomas are characterized by high levels of matrix metalloproteinase (MMP)-2 and MMP-9, which play crucial roles in tumor invasion and metastasis by degradation of the extracellular membrane leading to cancer cell spread to distal organs. We examined the effects of cytokines, mitogens, inducers and inhibitors on MMP-2 and -9 expression in osteosarcoma (U2OS) and rhabdomyosarcoma (RD). The selected compounds included natural cytokines and growth factors, as well as chemical compounds applied in therapy of sarcoma and natural compounds that have demonstrated anticancer therapeutic potential. These cell lines were cultured in their respective media to near confluence and the cells were washed with PBS and incubated in serum-free medium with various concentrations of several cytokines, mitogens and inhibitors. After 24 h the media were removed and analyzed for MMP-2 and -9 by gelatinase zymography and quantitated by densitometry. Osteosarcoma and rhabdomyosarcoma showed bands corresponding to MMP-2 and -9 with dose-dependent enhancement of MMP-9 with phorbol 12-myristate 13-acetate (PMA) treatment. Tumor necrosis factor-α, interleukin-1ß and LPS enhanced osteosarcoma U2OS MMP-9 secretion but had no effect on MMP-2 secretion. Tumor necrosis factor-α stimulated rhabdomyosarcoma MMP-2 expression, but had no effect on MMP-9 secretion. Doxycycline, epigallocatechin gallate, nutrient mixture (NM), actinomycin-D, cyclohexamide, retinoic acid and dexamethasone inhibited MMP-2 and -9 in U2OS osteosarcoma cells. PMA-treated RD cells showed dose-response inhibition of MMP-9 by doxycycline and epigallocatechin gallate and both MMPs by NM. Dexamethasone and actinomycin-D showed inhibition of MMP-2 secretion of RD cells. Our results show that cytokines, mitogens and inducers show variable upregulation of U2OS osteosarcoma and RD rhabdomyosarcoma MMP-2 and -9 secretion, and inhibitors demonstrate downregulation under stimulatory conditions, suggesting the application of these agents for the development of effective therapies in pediatric sarcomas.

In vitro modulation of MMP-2 and MMP-9 in adult human sarcoma cell lines by cytokines, inducers and inhibitors.

The highly aggressive adult sarcomas are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9, which play crucial roles in tumor invasion and metastasis by degradation of the extracellular membrane leading to cancer cell spread to distal organs. We examined the effect of cytokines, mitogens, inducers and inhibitors on MMP-2 and MMP-9 secretion in chondrosarcoma (SW-1353), fibrosarcoma (HT-1080), liposarcoma (SW-872) and synovial sarcoma (SW-982) cell lines. The selected compounds included natural cytokines and growth factors, as well as chemical compounds applied in therapy of sarcoma and natural compounds that have demonstrated anticancer therapeutic potential. MMP-2 and MMP-9 secretions were analyzed by gelatinase zymography following 24-h exposure to the tested agents and quantitated by densitometry. Fibrosarcoma, chondrosarcoma, liposarcoma and synovial sarcoma showed bands corresponding to MMP-2 and MMP-9 with dose-dependent enhancement of MMP-9 with phorbol 12-myristate 13-acetate (PMA) treatment. In chondrosarcoma cells, tumor necrosis factor (TNF)-α had a stimulatory effect on MMP-9 and insignificant effect on MMP-2 and interleukin (IL)-1ß stimulated MMP-9 and MMP-2. In fibrosarcoma and liposarcoma cells, TNF-α had a profound stimulatory effect on MMP-9, but no effect on MMP-2 and in synovial sarcoma an inhibitory effect on MMP-2 and no effect on MMP-9. IL-1ß had a slight inhibitory effect on fibrosarcoma, liposarcoma and synovial sarcoma MMP-2 and MMP-9 except for MMP-9 in synovial sarcoma which showed slight stimulation. Lipopolysaccharide (LPS) stimulated expression of MMP-2 in fibrosarcoma and chondrosarcoma while inhibited it in liposarcoma. Doxycycline, epigallocatechin gallate and the nutrient mixture inhibited MMP-2 and MMP-9 in all cell lines. Actinomycin-D, cyclohexamide, retinoic acid, and dexamethasone inhibited MMP-2 and -9 in chondrosarcoma and fibrosarcoma cells. Our results show that cytokines, mitogens, inducers and inhibitors have an up or down regulatory effect on MMP-2 and MMP-9 expression in adult sarcoma cell lines, suggesting these agents may be effective strategies to treat these cancers.

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.

Nutritional improvement of metabolic syndrome parameters in immature fructose-fed wild-type mice.

The incidence of type 2 diabetes mellitus is on the increase worldwide, with both environmental and genetic factors implicated in its development. Diabetes is often preceded by metabolic syndrome (MS) and may develop in normal adults ingesting a high fructose diet. The effect of high fructose intake on the development of MS in children and adolescents is less clear. Our objective was to study the effects of a nutrient mixture and metformin (MET), a widely used oral diabetic medication, in modulating the physiological and biochemical parameters of a high fructose diet in immature mice. C57BL/6J wild­type mice aged 7 weeks were administered 12% fructose in their water and MET in distilled water or a diabetic nutrient mix (DNM) over 7 weeks. DNM-fed mice showed a decrease in systolic blood pressure, total cholesterol and fructosamine compared to the fructose-fed only group (p<0.05). A discordant result was observed in the MET group, with a decrease in blood pressure but increases in total cholesterol and fructosamine (p<0.05). Serum glucose did not change significantly among the groups. Thus, symptoms of fructose-induced MS in young mice could be countered nutritionally. Additionally, MET may improve certain biochemical markers while worsening others.

Down-regulation of urokinase plasminogen activator and matrix metalloproteinases and up-regulation of their inhibitors by a novel nutrient mixture in human prostate cancer cell lines PC-3 and DU-145.

Strong clinical and experimental evidence shows that elevated levels of urokinase plasminogen activators (u-PA) and matrix metalloproteinases (MMPs) are associated with prostate cancer progression, metastasis and shortened survival in patients. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs). A nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract showed anticancer activity against a number of cancer cell lines. Our main objective was to study the effect of NM on the activity of u-PA, MMPs and their inhibitor TIMPs on human prostate cancer cell lines PC-3 and DU-145. Human prostate cancer cell lines PC-3 and DU-145 (ATCC) were grown in MEM media with 10% FBS and antibiotics in 24­well tissue culture plates. At near confluence, the cells were treated with NM at 0-1000 µg/ml in triplicate at each concentration. Analysis of u-PA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. Both PC-3 and DU-145 prostate cancer cell lines demonstrated u-PA activity (subunits 1 and 2, corresponding to 35 and 33 kDa). Prostate cancer cell line PC-3 secretion of u-PA subunit 1 was decreased by 65% at NM 500 µg/ml and subunit 2 by 100% at NM 50 µg/ml. Prostate cancer cell line DU-145 secretion of u-PA subunit 1 was decreased by 97% at NM 500 µg/ml and subunit 2 by 100% at NM 100 µg/ml. Untreated PC-3 showed two bands for MMP-2 and MMP-9. NM inhibited their expression in a dose-dependent manner. The activity of MMP-2 and MMP-9 was significantly inhibited at 250 µg/ml with total inhibition at 500 µg/ml. DU-145 cells did not exhibit MMP activity. Activity of TIMPs was up-regulated in both prostate cancer cell lines in a dose-dependent manner. Minimum activity was expressed at 50 µg/ml NM and maximum at 1000 µg/ml. Correlation analyses revealed a positive correlation between u-PA and MMPs and a negative correlation between u-PA/MMPs and TIMPs. These results suggest NM as a potential anticancer agent since it targets invasive parameters of prostate cancer.

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.

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 2.5×10(6) 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-1ß (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.