Formulation and characterization of EGCG for the treatment of superficial bladder cancer.

In the United States, the annual incidence of bladder cancer is approximately 70,000 new cases, with a mortality rate of approximately 15,000/year. The most common subtype (70%) of bladder cancer is superficial, namely hte non-muscle invasive disease form limited to the urothelium. The rate of progression and recurrence is up to 40 and 70%, respectively. Urothelial cell carcinoma of the bladder is typically treated with transurethral resection. The cancerous cells can float onto the adjacent epithelium, increasing the risk of recurrence. The standard of care is to offer adjuvant intravesical agents to reduce the risk of progression and recurrence. Current intravesical treatments are costly and are associated with special biohazard handling protocols. Patients are treated with intravesical therapy with bacillus Calmetter­Guerin (BCG) bacterium, or mitomycin C (MMC) following resection, both of which can cause moderate to severe side-effects which are rarely life-threatening. We previously examined the efficacy of epigallocatechin-3-gallate (EGCG) in comparison with MMC to prevent tumor cell implantation/growth in an animal model of superficial bladder cancer. Experiments revile that EGCG is slightly more effective than MMC at decreasing tumor cell implantation and consequent cancer growth in a bladder. This treatment requires the stringent sterile requirement of EGCG. EGCG can be unstable when sterilized at high temperatures. Thus, we evaluated two low temperature sterilization methods, such as ionizing radiation or the filtration method followed by freeze-drying. Both methods ensure the sterility of the sample; however, infrared and HPLC analysis revealed a slightly better stability of irradiated EGCG over the filtration method. The concentration of stable free radicals following irradiation was low, which are unlikely to exert any damaging effects to EGCG. Therefore, we consider that radiation will be the preferred method of EGCG sterilization, and that this may prove useful for the effective use of EGCG in the treatment of bladder cancer.

Effects of chlorhexidine, essential oils and herbal medicines (Salvia, Chamomile, Calendula) on human fibroblast in vitro.

Antiseptic rinses have been successfully used in inflammatory states of the gums and oral cavity mucosa. Antibacterial effects of chlorhexidine, essential oils and some herbs are well documented. Reaction of host tissue to these substances has much poorer documentation. The aim of the study was to analyse the influence of chlorhexidine (CHX), essential oil (EO: thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042%) mouth rinses and salvia, chamomile and calendula brews on fibroblast biology in vitro. The human fibroblast CCD16 line cells were cultured in incubation media which contained the examined substances. After 24 and 48 hours, the cell morphology, relative growth and apoptosis were evaluated. Exposure of fibroblasts to CHX, EO or salvia caused various changes in cell morphology. Cells cultured for 48 hours with CHX revealed a noticeably elongated shape of while cells cultured in high EO concentration or with salvia were considerably smaller and contracted with fewer projections. Chlorhexidine, EO and salvia reduced the fibroblast proliferation rate and stimulated cell death. Both reactions to EO were dose dependent. Cells exposure to chamomile or calendula brews did not change morphology or proliferation of fibroblasts. The results of this in vitro study showed that in contrast to chamomile and calendula, the brews of EO, CHX or salvia had a negative influence on fibroblast biology.

Unusual clotting dynamics of plasma supplemented with iron(III).

Iron salts are used in the treatment of iron deficiency anemia. Diabetic patients are frequently anemic and treatment includes administration of iron. Anemic patients on hemodialysis are at an increased risk of thromboembolic coronary events associated with the formation of dense fibrin clots resistant to fibrinolysis. Moreover, in chronic kidney disease patients, high labile plasma iron levels associated with iron supplementation are involved in complications found in dialyzed patients such as myocardial infarction. The aim of the present study was to investigate whether iron treatment is involved in the formation of the fibrin clots. Clotting of citrated plasma supplemented with Fe(3+) was investigated by thromboelastometry and electron microscopy. The results revealed that iron modifies coagulation in a complex manner. FeCl(3) stock solution underwent gradual chemical modification during storage and altered the coagulation profile over 29 days, suggesting that Fe(3+) interacts with both proteins of the coagulation cascade as well as the hydrolytic Fe(3+) species. Iron extends clotting of plasma by interacting with proteins of the coagulation cascade. Fe(3+) and/or its hydrolytic species interact with fibrinogen and/or fibrin changing their morphology and properties. In general FeCl(3) weakens the fibrin clot while at the same time precipitating plasma proteins immediately after application. Fe(3+) or its derivatives induced the formation of insoluble coagulums in non-enzymatic reactions including albumin and transferrin. Iron plays a role in coagulation and can precipitate plasma proteins. The formation of coagulums resistant to lysis in non­enzymatic reactions can increase the risk of thrombosis, and extending clotting of plasma can prolong bleeding.

Analysis of the inhibition of PAI-1 by metal theaflavin complexes and their degradation products.

The inhibition of elements of the plasminogen activator system [urokinase (uPA), tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1)] plays an important role in human diseases. PAI-1 is overexpressed in obesity and diabetes, and the inhibition of this protein has been postulated to alleviate the symptoms of both disorders. We found that two theaflavins (TFs) from black tea inhibit PAI-1 and we suggest that the beneficial effects of drinking tea may be associated with the suppression of PAI-1 activity by theaflavins. Epidemiological studies are controversial; some studies show the beneficial effects of drinking black tea on obesity and diabetes, while others do not. TFs, a family of compounds that can comprise up to 40% the dry weight of black tea, are responsible for the characteristic color, and they are known to chelate metals. We hypothesized that the content/variety of metals present in drinking water may be one of the reasons for such controversies in the population studies. TFs are excellent chelating compounds by trapping metals into complexes; thus, the quality of water used for tea preparation may influence changes in the formation of new products according to TF affinity for different metals, as well as their high molecular weight oxidation products. Our modeling and docking studies suggest that TF/metal complexes have similar affinity to PAI-1 as native TFs. However, analyses using liquid chromatography-mass spectroscopy (LC-MS) revealed the presence of TF degradation products in tea brewed using water containing metal salts. These can further form high molecular weight oxidation products. Thus, metals present in tea could diminish the beneficial effects of black tea by reducing TF concentration via metal-induced degradation and precipitation.

A study of the anti-diabetic agents of camel milk.

The number of people diagnosed with type 2 diabetes has risen steeply recently exhausting the ability of health care systems to deal with the epidemic. Seventy-five percent of people with diabetes live in low- and middle-income countries. The largest populations of diabetics are in China and India, with many of those people living in extreme poverty. Combined forces of governmental health care, charities and donation of pharmaceutical companies would not be able to cope with the financial demands needed for medicaments and treatments for these people. Therefore, it is worth looking into traditional folk remedies to find if there is any scientific merit to justify their claims for alleviating symptoms of diabetes. There is a traditional belief in the Middle East that regular consumption of camel milk helps in the prevention and control of diabetes. Recently, it has been reported that camel milk can have such properties. Literature review suggests the following possibilities: i) insulin in camel milk possesses special properties that makes absorption into circulation easier than insulin from other sources or cause resistance to proteolysis; ii) camel insulin is encapsulated in nanoparticles (lipid vesicles) that make possible its passage through the stomach and entry into the circulation; iii) some other elements of camel milk make it anti-diabetic. Sequence of camel insulin and its predicted digestion pattern do not suggest differentiability to overcome the mucosal barriers before been degraded and reaching the blood stream. However, we cannot exclude the possibility that insulin in camel milk is present in nanoparticles capable of transporting this hormone into the bloodstream. Although, much more probable is that camel milk contains 'insulin-like' small molecule substances that mimic insulin interaction with its receptor.

Diverse inhibition of plasminogen activator inhibitor type 1 by theaflavins of black tea.

Fruits, vegetables, spices and a variety of teas are suggested for the prevention of many diseases. They encompass active, non-nutritional ingredients called nutraceuticals which are defined as food products that provide health benefits. Many nutraceuticals have been tested to identify inhibitors of plasminogen activator inhibitor (PAI-1). PAI-1 is the major and fast acting physiological inhibitor of fibrinolysis. However, preclinical studies of PAI-1 inhibitors have revealed an additional role of PAI-1 in the pathogenesis of vascular remodeling, renal injury, diabetes, obesity, Alzheimer's disease and cancer. Thus PAI-1 is a potential therapeutic target in some of these diseases. Our previous study revealed that a black tea extract (containing mostly theaflavins) inhibits PAI-1. In this study we report results for four pure (>98%) theaflavins. Inactivation of PAI-1 was tested by clot formation and by its lysis using thromboelastometry and measurements of human plasma turbidity. Among four tested theaflavins, theaflavin-3'-gallate was the most potent in PAI-1 inhibition trailed by theaflavin-3,3'-digallate, while the other two i.e., theaflavin and theaflavin-3-gallate did not show inhibitory activity.

Theaflavin digallate inactivates plasminogen activator inhibitor: could tea help in Alzheimer's disease and obesity?

Proteolysis in general and particularly the serine proteases are causally involved in many physiological processes and different diseases. Recently it was reported that plasminogen activator inhibitor type one (PAI-1) inactivation can alleviate the symptoms of Alzheimer's disease and reduce the body weight of obese individuals. In our broad search for natural compounds and their derivatives that can inhibit PAI-1, we include the polyphenols of teas since teas (green and black) or their components have been reported to alleviate the symptoms of both obesity and Alzheimer's disease. Inactivation of PAI-1 was measured in human plasma using thromboelastography. We used known PAI-1 inhibitor PAI039 [{1-benzyl-5-[4-(trifluoromethoxy) phenyl]-1H-indol-3-yl}(oxo)acetic acid] as a positive control and (-)-epigallo-catechin-3-gallate (EGCG), its prodrug octaacetate EGCG (OcAc EGCG) and theaflavin digallate [TH(2)] as potential PAI-1 inhibitors. We found that inactivation of PAI-1 in plasma by EGCG and OcAc EGCG was low or very low. However, TH(2) inactivated PAI-1 in a concentration-dependent manner with an IC50 of 18 microM which is equal to or better than the IC50 reported for known PAI-1 inhibitor PAI039. Clearly TH(2) inhibits PAI-1 and might play a role in slowing down the progression of Alzheimer's disease or obesity by a PAI-1-dependent pathway. While the clinical value of TH(2) has not been proven, long-term prospective studies assessing its efficacy are warranted due to the benign nature of the substance.

Yin and yang of the plasminogen activator inhibitor.

Plasminogen activator inhibitor type one (PAI-1) is involved in many biological processes, and depending on a disease its overexpression, deficiency or normal level can sometimes produce surprising outcomes. This paper reviews low, high, and normal levels of PAI-1 in relation to diseases, their incidents, and possible treatment. Also, a role of PAI-1 in cancer is discussed with attention to "PAI-1 paradox" and explanation of the possible therapeutic applications using this protein.

Nutraceutical inhibitors of urokinase: potential applications in prostate cancer prevention and treatment.

Epidemiological studies have shown that the clinical incidence of prostate cancer varies by geographical area. When individuals move from low to high prostate cancer incidence areas, the risk of developing cancer increases to the level observed in the indigenous population. It was hypothesized that this observation is related to diet or more specifically to nutraceuticals present in food, medicinal plants, and herbs. Nutraceuticals can inhibit or downregulate enzymes critical for cancer formation. We tested this hypothesis by searching the 3D database of nutraceuticals and docking them to the 3D structure of urokinase. In addition to nutraceuticals, the data-base contains known uPA inhibitors that served as positive controls. From >1,000 compounds, several potential uPA inhibitors have been selected (antipain, leupeptin, folic acid, rosmarinic acid, lavendustin A, fisetin, myricetin, tolfenamic acid). Some of these were subject to further tests on inhibitory activity and inhibition of sprout formation. We found that compounds selected by computational methods indeed inhibit uPA and sprout formation. However, because the database of nutraceuticals was small, we did not expect to find either many or high affinity/specific inhibitors. Rather, we tested this method as a proof of concept. All the facts described above support the hypothesis that nutrients selected by computerized searches can inhibit unwanted uPA activity and thus reduce angiogenesis. If true, a proper diet rich in uPA-inhibiting nutraceuticals might support the prevention of prostrate cancer and be a supportive tool in prostate cancer treatment.