Polyphenols, found abundantly in plants, display many anticarcinogenic properties including their inhibitory effects on cancer cell proliferation, tumor growth, angiogenesis, metastasis, and ...inflammation as well as inducing apoptosis. In addition, they can modulate immune system response and protect normal cells against free radicals damage. Most investigations on anticancer mechanisms of polyphenols were conducted with individual compounds. However, several studies, including ours, have indicated that anti-cancer efficacy and scope of action can be further enhanced by combining them synergistically with chemically similar or different compounds. While most studies investigated the anti-cancer effects of combinations of two or three compounds, we used more comprehensive mixtures of specific polyphenols and mixtures of polyphenols with vitamins, amino acids and other micronutrients. The mixture containing quercetin, curcumin, green tea, cruciferex, and resveratrol (PB) demonstrated significant inhibition of the growth of Fanconi anemia head and neck squamous cell carcinoma and dose-dependent inhibition of cell proliferation, matrix metalloproteinase (MMP)-2 and -9 secretion, cell migration and invasion through Matrigel. PB was found effective in inhibition of fibrosarcoma HT-1080 and melanoma A2058 cell proliferation, MMP-2 and -9 expression, invasion through Matrigel and inducing apoptosis, important parameters for cancer prevention. A combination of polyphenols (quercetin and green tea extract) with vitamin C, amino acids and other micronutrients (EPQ) demonstrated significant suppression of ovarian cancer ES-2 xenograft tumor growth and suppression of ovarian tumor growth and lung metastasis from IP injection of ovarian cancer A-2780 cells. The EPQ mixture without quercetin (NM) also has shown potent anticancer activity in vivo and in vitro in a few dozen cancer cell lines by inhibiting tumor growth and metastasis, MMP-2 and -9 secretion, invasion, angiogenesis, and cell growth as well as induction of apoptosis. The presence of vitamin C, amino acids and other micronutrients could enhance inhibitory effect of epigallocatechin gallate (EGCG) on secretion of MMPs. In addition, enrichment of NM with quercetin (EPQ mix) enhanced anticancer activity of NM in vivo. In conclusion, polyphenols, especially in combination with other polyphenols or micronutrients, have been shown to be effective against multiple targets in cancer development and progression, and should be considered as safe and effective approaches in cancer prevention and therapy.
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 5×105 B16FO murine melanoma cells or to injection of 5×105 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.
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-1β 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-1β 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.
The prognosis of hepatocellular carcinoma (HCC) remains dismal despite any treatment. Matrix metalloproteinases (MMPs) have been researched for their role in tumor invasion and metastasis. Various ...cytokines, mitogens, growth factors, inducers, and inhibitors control MMP activities. In this article, we investigated the roles of these in the regulation of MMP-2, -9 secretions in HCC. Human HCC SK-Hep-1 was grown in appropriate media. At near confluence, the cells were washed with phosphate-buffered saline and incubated in serum-free media with PMA; TNF-α, IL-1β; lipopolysaccharide; epigallocatechin gallate (EGCG) and doxycycline (Dox) at various doses with and without PMA; a nutrient mixture (NM) containing lysine, proline, ascorbic acid, and EGCG with and without PMA at; and actinomycin D and cycloheximide at different doses. After 24 hours, the media were removed and analyzed. SK-Hep-1 expressed bands corresponding to MMP-2 and MMP-9. TNF-α showed an insignificant effect on MMP-2 at doses below 25 at which dose MMP-2 was virtually blocked and a moderate dose-dependent effect on MMP-9. Interleukin-1β demonstrated an insignificant effect on MMP-2 at doses below 25 at which dose MMP-2 was completely blocked and enhanced effects on MMP-9. Lipopolysaccharide showed dose-dependent inhibition of MMP-2 and MMP-9. EGCG, Dox, and NM, without and with PMA, downregulated the expression of MMP-2 and MMP-9. Actinomycin D and cycloheximide also had dose-dependent inhibitory effects on MMPs. Our results showed that cytokines, mitogens, and inhibitors modulated SK-Hep-1 MMP-2 and MMP-9 secretion, suggesting the clinical use of especially potent and nontoxic MMP inhibitor as the NM in management of HCC.
Ovarian cancer is the deadliest gynecological malignancy in women, and fifth leading cause of death. Despite advances made in chemotherapy and surgery, the average time of clinical remission is ...approximately 2 years and the 5-year survival rate is 45%. Thus, there is an urgent need for the development of a novel therapeutic approach to ovarian cancer treatment. We investigated the effect of a specific nutrient mixture (EPQ) containing ascorbic acid, lysine, proline, green tea extract, and quercetin on human ovarian cancer cell A-2780 in vivo and in vitro. Athymic female nude mice (n = 12) were all inoculated intraperitoneally (IP) with 2 × 10⁶ cells in 0.1 mL of phosphate buffered saline (PBS) and randomly divided into two groups. Upon injection, the Control group (n = 6) was fed a regular diet and the EPQ group (n = 6) a regular diet supplemented with 0.5% EPQ. Four weeks later, the mice were sacrificed and tumors that developed in the ovary were excised, weighed, and processed for histology. Lungs were inspected for metastasis. In vitro, A-2780 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% FBS and antibiotics. At near confluence, cells were treated with EPQ in triplicate at concentrations between 0 and 1000 μg/mL. Cell proliferation was measured via MTT assay, MMP-9 secretion via gelatinase zymography, invasion through Matrigel and morphology via hematoxylin and eosin (H & E) staining. All Control mice developed large ovarian tumors, whereas 5 out of 6 mice in the EPQ group developed no tumors, and one, a small tumor. Control mice also showed lung metastasis in 6 out of 6 mice, while no lung metastasis was evident in EPQ mice. Zymography demonstrated only MMP-9 expression, which EPQ inhibited in a dose-dependent fashion, with virtual total block at 250 μg/mL concentration. EPQ significantly inhibited invasion through Matrigel with total block at 250 μg/mL concentration. MTT showed dose-dependent inhibition of cell proliferation with EPQ, and H & E staining showed no morphological changes below 500 μg/mL EPQ. These results suggest that EPQ has therapeutic potential in the treatment of ovarian cancer by significantly suppressing ovarian tumor incidence and growth and lung metastasis, and by inhibiting MMP-9 secretion and invasion of A-2780 ovarian cancer cells.
The limited ability of current treatments to control metastasis and the proposed antitumor properties of specific nutrients prompted us to examine the effect of a specific formulation (nutrient ...supplement NS) of lysine, proline, arginine, ascorbic acid, and green tea extract in vivo on the development of N-methyl-N-nitrosourea (MNU)-induced mammary tumors in rats.
A single intraperitoneal dose of MNU was injected into each of 20 female Sprague-Dawley rats (aged 50 days) to induce tumors. Two weeks after MNU treatment, a time by which the animals had recovered from MNU-induced toxicity, the rats were divided into two groups. Rats in group 1 (n = 10) were fed Purina chow diet, whereas those in group 2 (n = 10) were fed the same diet supplemented with 0.5% NS. After a further 24 weeks, the rats were killed and tumors were excised and processed.
NS reduced the incidence of MNU-induced mammary tumors and the number of tumors by 68.4%, and the tumor burden by 60.5%. The inhibitory effect of NS was also reflected by decreased tumor weight; the tumor weights per rat and per group were decreased by 41% and 78%, respectively. In addition, 30% of the control rats developed ulcerated tumors, in contrast to 10% in the nutrient supplemented rats.
These findings suggest that the specific formulation of lysine, proline, arginine, ascorbic acid, and green tea extract tested significantly reduces the incidence and growth of MNU-induced mammary tumors, and therefore has strong potential as a useful therapeutic regimen for inhibiting breast cancer development.
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)-1β 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-1β 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.
The incidence of hepatocellular carcinoma (HCC), once thought to be a rare tumor in North America, has rapidly increased in recent years in the United States. Current treatment modalities to halt the ...progression of this disease are only marginally effective. The mainstay treatment is liver transplantation, which is often confronted with donor shortage. Invasion, metastasis and recurrence contribute to the high mortality rate of this disease. Matrix metalloproteinases (MMPs) that degrade the extracellular matrix (ECM) have been associated with the progression, invasion and metastasis of the disease. We have developed strategies to strengthen the ECM collagen and inhibit MMPs through micronutrients such as lysine, proline and ascorbic acid. Addition of epigallocatechin gallate or green tea extract to these micronutrients synergistically enhanced anti-carcinogenic activity in HepG2 cells. Addition of certain other micronutrients, such as N-acetylcysteine, selenium, copper and zinc (NM) synergistically enhanced the anticancer activity of the mixture in a model of hepatocellular carcinoma using HepG2 cells. In vitro studies using HepG2 demonstrated that NM was very effective in inhibiting cell proliferation (by MTT assay), MMPs secretion (by gelatinase zymography), cell invasion (through Matrigel) and induction of apoptosis (by live green caspase). In addition, NM was shown to down-regulate urokinase plasminogen activator (by fibrin zymography) and up-regulate tissue inhibitors of metalloproteinases (by reverse zymography) in another HCC cell line, SK-Hep-1. MMP-2 and MMP-9 activities were further modulated by phorbol 12-myristate 13-acetate (PMA) induction and inhibited by NM. In previous studies, NM inhibited Sk-Hep-1 xenografts in nude mice and also inhibited hepatic metastasis of B16FO melanoma cells. Our results suggest that NM is an excellent candidate for therapeutic use in the treatment HCC by inhibiting critical parameters in cancer development and progression, such as proliferation, invasion and metastasis, and by inducing apoptosis.
Background: Matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), Ki 67 (proliferative protein) and
constituents of ECM play a critical role in angiogenesis, and are crucial in ...neoplastic invasion and metastasis. Based on
the antitumor properties of certain nutrients, we investigated the effect of a diet containing lysine, proline, arginine,
ascorbic acid and green tea extract on the growth of tumors induced by implanting human prostate cancer PC-3 cells in athymic
nude mice and on the expression of MMPs, VEGF, Ki 67 and fibronectin in these tumors, as well as the production of mucin (by
PAS staining). Materials and Methods: Male nude mice (n=12) were inoculated with 3Ã10 6 prostate cancer PC-3 cells and randomly divided into two groups; Group A was fed a regular diet and Group B was fed a regular
diet supplemented with 0.5% of the nutrient mixture (NM). Four weeks later, tumors were excised, weighed and processed for
histology. Results: The results showed inhibition of tumor growth in Group B. Histological studies revealed inhibition of
MMP-9 and VEGF secretion and mitosis in Group B tissues. Conclusion: Nutrient supplementation strongly suppressed the growth
of tumors without any adverse effects in nude mice, suggesting strong potential as an anticancer agent.
Aims: Bladder cancer, the fourth highest incident cancer in men and tenth in women, is associated with a high rate of recurrence, even when treated in situ, and prognosis is poor once the cancer ...metastasizes to distant sites. Based on anticancer properties, we investigated the effect of a mixture of lysine, proline, arginine, ascorbic acid, and green tea extract on human bladder cancer cells T‐24 by measuring: proliferation, matrix metalloproteinase (MMP) expression, and cancer cell invasive potential.
Methods: Human bladder cancer cells T‐24 (ATCC) were grown in McCoy medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 mg/mL) in 24‐well tissue culture plates. At near confluence, the cells were treated with the nutrient mixture dissolved in media and tested at 0, 10, 50, 100, 500, and 1000 µg/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 activity by gelatinase zymography, and invasion through Matrigel.
Results: Nutrient mixture inhibited the T‐24 cell secretion of MMP‐2 and ‐9, with virtual total inhibition of MMP‐2 at 500 µg/mL and MMP‐9 at 100 µg/mL. The nutrient mixture significantly reduced the invasion of human bladder cancer cells T‐24 through Matrigel in a dose‐dependent fashion, with 95% inhibition at 500 µg/mL and 100% at 1000 µg/mL nutrient mixture (P < 0.001).
Conclusion: Our results suggest that our nutrient mixture is an excellent candidate for therapeutic use in the treatment of bladder cancer, by inhibiting critical steps in cancer development and spread, such as MMP secretion and invasion.
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