Original Article Hypoascorbemia induces atherosclerosis and vascular deposition of lipoprotein(a) in transgenic mice

Abstract: Lipoprotein(a), a variant of LDL carrying the adhesive glycoprotein apo(a), is a leading risk factor for cardiovascular disease. Lipoprotein(a) (Lp(a)) is found in humans and subhuman primates but rarely in lower mammals. Better understanding of the evolutionary advantage of this molecule should elucidate its physiological role. We developed a new mouse model with two characteristics of human metabolism: the expression of Lp(a) and the lack of endogenous ascorbate (vitamin C) production. We show that dietary deficiency of ascorbate increases serum levels of Lp(a). Moreover, chronic hypoascorbemia and complete depletion of ascorbate (scurvy) leads to Lp(a) accumulation in the vascular wall and parallels atherosclerotic lesion development. The results suggest that dietary ascorbate deficiency is a risk factor for atherosclerosis independent of dietary lipids. We provide support for the concept that Lp(a) functions as a mobile repair molecule compensating for the structural impairment of the vascular wall, a morphological hallmark of hypoascorbemia and scurvy

1323-1333

Abstract. 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

Modulation of N-methyl-N-nitrosourea induced mammary tumors in Sprague–Dawley rats by combination of lysine, proline, arginine, ascorbic acid and green tea extract

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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

Anticancer Efficacy of Polyphenols and Their Combinations

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

Inhibition of <i>Borrelia Burgdorferi</i>-Induced TLR2-NFκB Canonical Signaling by Gallic Acid through Targeting the CD14+ Adaptor Protein and p65 Molecule

The cases of Lyme disease caused by Borrelia burgdorferi infection have been increasing throughout Northern America and Europe. This pathogen, if not treated in a timely manner with antibiotics, can cause persisting and debilitating health outcomes. In the search for novel agents against B. burgdorferi, we investigated a phenolic compound—gallic acid—for its anti-Borrelia and anti-inflammatory effects. Our results showed its biocidal effect starting from 100 μg/mL against active spirochetes, persisters/round-shaped bodies, and biofilm like aggregates of B. burgdorferi sensu stricto. Activation of macrophages by live B. burgdorferi also resulted in a robust NFκB-dependent proinflammatory responses seen in increased production of cytokines. Using human CD14+ macrophages in vitro, we showed that CD14+ adaptor and phosphorylated p65 molecule are impeded at nonbiocidal and noncytotoxic concentrations of gallic acid, resulting in the inhibition of both expression and secretion of cytokines IL1β, IL6, and TNFα. Our findings demonstrate efficacy of gallic acid against B. burgdorferi and provide potential mechanistic insight into its TLR2/CD14+-NFκB mediated mode of action. Further studies on the potential of gallic acid as a safe and effective compound against Borrelia-caused infection are warranted

Anti-borreliae efficacy of selected organic oils and fatty acids

Abstract Background Borrelia sp. is a causative pathogen of Lyme disease which has become a worldwide health concern. Non-toxic approaches especially directed toward latent persistent forms of this pathogen are desired. Lipids in the form of volatile and non-volatile oils, and fatty acids with proven anti-borreliae efficacy could become an additional support or an alternative for consideration in treatment approaches. Methods In this study we investigated 47 lipids (30 volatile and non-volatile oils, and 17 fatty acids) of plant and animal origin against typical motile, knob/round-shaped persisters, and biofilm-like aggregates of Borrelia burgdorferi s.s. and Borrelia garinii, which are identified as pathogenic factors of Lyme disease in the USA and Europe, using direct microscopic counting and spectrofluorometric measurements. Results Out of all examined lipids, 5 oils (Bay leaf oil, Birch oil, Cassia oil, Chamomile oil German, and Thyme oil) at or below 0.25%, and 3 fatty acids (13Z,16Z Docosadienoic acid, erucic acid, and petroselinic acid) at or below 0.75 mg/ml, showed bactericidal activity against typical motile spirochetes and knob/round-shaped persisters. Only Bay leaf oil and Cassia oil, including their major constituents, eugenol and cinnamaldehyde, showed to target biofilm-like aggregates of both tested Borrelia spp. at the same concentration, although with 20–30% eradication mark. Conclusion Based on obtained results, volatile oils were more potent than non-volatile oils, and unsaturated fatty acids were more effective than saturated fatty acids. Among all tested oils, Bay leaf oil and Cassia oil, with their major components eugenol and cinnamaldehyde, seem to have the highest anti-borreliae efficacy

10-undecynoic acid is a new anti-adherent agent killing biofilm of oral Streptococcus spp.

In the search for novel agents against oral pathogens in their planktonic and biofilm form, we have focused our attention on 10-undecynoic acid as the representative of the acetylenic fatty acids. Using macro-broth susceptibility testing method we first established MIC value. Next, the MBC value was determined from a broth dilution minimum inhibitory concentration test by sub-culturing it to BHI agar plates that did not contain the test agent. Anti-biofilm efficacy was tested in 96-well plates coated with saliva using BHI broth supplemented with 1% sucrose as a standard approach. Based on obtained results, MIC value for 10-undecynoic acid was established to be 2.5 mg/ml and the MBC value to be 5 mg/ml. The MBIC90 showed to be 2.5 mg/ml, however completed inhibition of biofilm formation was achieved at 5.0 mg/ml. MBBC concentration revealed to be the same as MBC value, causing approximately 30% reduction at the same time in biomass of pre-existing biofilm, whereas application of 7.0 mg/ml of 10-undecynoic acid crossed the 50% eradication mark. Strong anti-adherent effect was observed upon 10-undecynoic acid application at sub-MBC concentrations as well, complemented with suppression of acidogenicity and aciduricity. Thus, we concluded that 10-undecynoic acid might play an important role in the development of alternative or adjunctive antibacterial and anti-biofilm preventive and/or therapeutic approaches

A Novel Nutrient Mixture Induces Apoptosis in Human Mesothelioma Cells (MSTO-211H) via Activation of Caspases

Background: Malignant mesothelioma is a highly aggressive and fatal cancer of older people. Consistently associated with asbestos exposure, mesothelioma is diagnosed when it is extensively metastasized and it has a dismal prognosis.&nbsp;Purpose of the study: Surgery, chemotherapy, and radiotherapy are the mainstay of treatment yet they are ineffective in increasing the mesothelioma patient survival. Therefore, a different approach is needed. A novel nutrient mixture containing green tea extract, ascorbic acid, lysine, and proline exhibited anti-cancer effects in various cancers. In our previous studies, the nutrient mixture was seen to block MMP secretion and invasion through MatrigelTM by mesothelioma cells. In the current study, we wanted to explore if the nutrient mixture could induce apoptosis in mesothelioma cells.&nbsp;Results and main findings: The mesothelioma MSTO-211H cells were cultured in complete Ham F-12K medium and the cells were then treated with the nutrient mixture at 0-1000 µg/ml concentration. Cell cytotoxicity was measured by MTT assay, morphology by H&amp;E staining, and the apoptosis by LIVE Green Caspases. The nutrient mixture did not show significant inhibition of cell proliferation. However, H&amp;E staining at the dose of 100 µg/ml showed a few cellular changes, while significant changes pertaining to apoptosis morphology were observed at 500 and 1000 µg/ml. LIVE Green Caspases analysis showed cells in early an d late apoptosis with increasing doses of the nutrient mixture.&nbsp;Conclusions: Our results suggest that the nutrient mixture may provide a new supplemental strategy for enhancing the therapeutic options for mesothelioma</p