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.
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 OHSU‑974 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.
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.
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)-1β 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-1β 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.
MMP-2 and MMP-9 secretion is elevated in several types of human cancers and their elevated expression has been associated with poor prognosis. Expression of MMPs is highly regulated by cytokines and ...signal transducation pathways, including those activated by phorbol 12-myristate 13-acetate (PMA). The aim of this study was to examine the effect of PMA on MMP-2 and MMP-9 secretion in 42 different human cancer cell lines, selected on the basis of their organ malignancies. They were cultured in the recommended media supplemented with 10% FBS and antibiotics in 24-well tissue culture plates. At near confluence, the cells were washed with PBS, 0.5 ml of medium was added, and the cultures were incubated. Parallel sets of cultures were also treated with PMA for induction of enzymes. After 24 h the media were collected and MMP-2 and MMP-9 levels were assayed by gelatinase zymography. Based on MMP-2 and MMP-9 secretion without and with PMA treatment, the various human cancer cell lines fell into one of two major groups. The first group characterized by low basal MMP-9 secretion fell into three different categories of susceptibility to PMA induction of MMP-9 expression: resistant, moderately susceptible and highly susceptible. High basal MMP-9 levels responsive to PMA induction characterized the second group. Most cancer cell lines examined exhibited basal levels of MMP-2, MMP-9 or both. MMP-2 secretion was not induced by PMA in any of the cancer cells examined.
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-1β 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.
Consumption of a plant-based diet has been associated with prevention of the development and progression of cancer. We have developed strategies to inhibit cancer development and its spread by ...targeting common mechanisms used by all types of cancer cells that decrease stability and integrity of connective tissue. Strengthening of collagen and connective tissue can be achieved naturally through the synergistic effects of selected nutrients, such as lysine, proline, ascorbic acid and green tea extract (NM). This micronutrient mixture has exhibited a potent anticancer activity
in vivo
and
in vitro
in a few dozen cancer cell lines. Its anti-cancer effects include inhibition of metastasis, tumor growth, matrix metalloproteinase (MMP) secretion, invasion, angiogenesis, and cell growth as well as induction of apoptosis. Many cancers are often diagnosed at later stages, when metastasis has occurred, which standard treatment has been unable to control. Our studies on NM effects on hepatic and pulmonary metastasis demonstrated profound, significant suppression of metastasis in a murine model. Evaluation of effects of NM on xenografts in murine models demonstrated significant reduction in tumor size and tumor burden in all human cancer cell lines tested.
In vitro
studies demonstrated that NM was very effective in inhibition of cell proliferation (by MTT assay), MMP secretion (by gelatinase zymography), cell invasion (through Matrigel), cell migration (by scratch test), induction of apoptosis (by live green caspase) and induction of pro-apoptotic genes in many diverse cancer cell lines. Furthermore,
in vivo
and
in vitro
studies of effects of individual micronutrients compared to their specific combination demonstrated synergistic effects resulting in improved anticancer potency.
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.
Uterine leiomyosarcoma is a rare malignant smooth muscle tumor originating in the uterine wall that generally responds poorly to chemotherapy and radiation.
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.
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 • 10
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.
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.
The results suggest a therapeutic potential for nutrient mixture in uterine leiomyosarcoma treatment.
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 phosphate‑buffered 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-1β (IL-1β) 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-1β 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.
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