Can EGCG Alleviate Symptoms of Down Syndrome by Altering Proteolytic Activity?

Down syndrome (DS), also known as "trisomy 21", is a genetic disorder caused by the presence of all or part of a third copy of chromosome 21. Silencing these extra genes is beyond existing technology and seems to be impractical. A number of pharmacologic options have been proposed to change the quality of life and lifespan of individuals with DS. It was reported that treatment with epigallocatechin gallate (EGCG) improves cognitive performance in animal models and in humans, suggesting that EGCG may alleviate symptoms of DS. Traditionally, EGCG has been associated with the ability to reduce dual specificity tyrosine phosphorylation regulated kinase 1A activity, which is overexpressed in trisomy 21. Based on the data available in the literature, we propose an additional way in which EGCG might affect trisomy 21-namely by modifying the proteolytic activity of the enzymes involved. It is known that, in Down syndrome, the nerve growth factor (NGF) metabolic pathway is altered: first by downregulating tissue plasminogen activator (tPA) that activates plasminogen to plasmin, an enzyme converting proNGF to mature NGF; secondly, overexpression of metalloproteinase 9 (MMP-9) further degrades NGF, lowering the amount of mature NGF. EGCG inhibits MMP-9, thus protecting NGF. Urokinase (uPA) and tPA are activators of plasminogen, and uPA is inhibited by EGCG, but regardless of their structural similarity tPA is not inhibited. In this review, we describe mechanisms of proteolytic enzymes (MMP-9 and plasminogen activation system), their role in Down syndrome, their inhibition by EGCG, possible degradation of this polyphenol and the ability of EGCG and its degradation products to cross the blood-brain barrier. We conclude that known data accumulated so far provide promising evidence of MMP-9 inhibition by EGCG in the brain, which could slow down the abnormal degradation of NGF.

Can components of the plasminogen activation system predict the outcome of kidney transplants?

Proteolytic and antiproteolytic enzymes play a critical role in the physiology and pathology of different stages of human life. One of the important members of the proteolytic family is the plasminogen activation system (PAS), which includes several elements crucial for this review: the 50 kDa glycoprotein plasminogen activator inhibitor 1 (PAI-1) that inhibits tissue-type (tPA) and urokinase-type plasminogen activator (uPA). These two convert plasminogen into its active form named plasmin that can lyse a broad spectrum of proteins. Urokinase receptor (uPAR) is the binding site of uPA. This glycoprotein on the cell surface facilitates urokinase activation of plasminogen, creating high proteolytic activity close to the cell surface. PAS activities have been reported to predict the outcome of kidney transplants. However, reports on expression of PAS in kidney transplants seem to be controversial. On the one hand there are reports that impaired proteolytic activity leads to induction of chronic allograft nephropathy, while on the other hand treatment with uPA and tPA can restore function of acute renal transplants. In this comprehensive review we describe the complexity of the PAS as well as biological effects of the PAS on renal allografts, and provide a possible explanation of the reported controversy.

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.

Synergistic anticancer activity of biologicals from green and black tea on DU 145 human prostate cancer cells.

There is considerable interest in the potential of botanicals in preventing and/or alleviating chronic ailments. Among the most studied botanicals are compounds present in green and black teas. Nontoxic tea polyphenols are potent antioxidants, and they also modulate several signalling pathways and inhibit proteins such as MMP-9 or protein plasminogen activator system, making them very attractive potential therapeutics. One criticism of the prophylactic or therapeutic use of green or black tea polyphenols was presumably the poor bioavailability of these chemicals when ingested. However, studies have shown that epigallocatechin-3-gallate (EGCG) and theaflavin (TF) can be detected in the small and large intestine, liver, and prostate of experimental animals after consumption of tea extracts. In particular, a study was carried out on 20 men scheduled for prostatectomy, who were assigned to consume teas for five days before surgery. Tea polyphenols were detected in the prostate. This fact contradicts the common misconception of poor bioavailability of TF and EGCG and makes feasible the application of green or black tea polyphenols as prophylactic and therapeutic agents. Theaflavins and catechins seem to act on cancer cells largely through different pathways, so utilisation of both could offer synergistic anticancer effects, but so far no work has been done on the cumulative effects of EGCG and TF on prostate cancer. Therefore, in this study we have investigated if EGCG in combination with TF can reduce the rate of prostate cancer growth, and we have observed greater cell death compared to application of either TF or EGCG alone.

The concentration of 12-lipoxygenase in platelet rich plasma as an indication of cancer of the prostate.

AIM OF THE STUDY: The aim of this study was to determine whether measuring concentrations of 12-LOX in platelet-rich plasma patients can:Differentiate between the group of patients with prostate cancer and healthy men.Correlate the degree of severity of the disease and the concentration of the enzyme. MATERIAL AND METHODS: The study group comprised 88 men (40-88 years), including 54 patients diagnosed with prostate cancer. The population was divided into 4 groups:group 1 (22 men, aged 55-84 years) -with a negative biopsy,group 2 (36 men, aged 54-88 years) - with a positive biopsy result,group 3 (18 participants aged 58-83) - patients with cancer metastatic disease,group 4 of healthy men (12 people aged 40-66 years) - biopsy was not performed. Routine PSA, morphology and CRP analysis were performed and platelet rich plasma was used for 12(S)LOX determination using an ELISA kit. RESULTS: There was a weak (r = 0.0487) positive correlation between the number of blood platelets and plasma 12(S)LOX.An inverse relationship between 12(S)LOX and Gleason grade was found.Heterogeneity of 12(S)LOX in the group with prostate cancer metastatic disease may suggest differences in the response to the treatment carried out.There were no statistically significant differences in concentrations of 12(S)LOX in different groups of patients. CONCLUSIONS: Our results suggest that 12(S)LOX is relevant in prostate cancer; however, further study should include a larger, more select group of men with prostate cancer.

Very long half-life plasminogen activator inhibitor type 1 reduces bleeding in a mouse model.

OBJECTIVE: To investigate the potential for the future clinical use of a very long half-life plasminogen activator inhibitor type 1 (VLHL PAI-1) as a haemostatic agent. MATERIALS AND METHODS: We developed a VLHL PAI-1 (half-life >700 h) recombinant mutant of PAI-1 and assessed VLHL PAI-1 for its ability to inhibit fibrinolysis in vitro using human, rabbit, mouse and rat blood. Fibrin clot lysis time, monitored by thromboelastometry, was determined at various concentrations of VLHL PAI-1. Also, we determined total bleeding time and total blood loss of control, VLHL PAI-1-, tissue-type plasminogen activator (tPA)- and tPA + VLHL PAI-1-treated mice. RESULTS: Using a thromboelastometer, mouse blood was most similar to human blood in its coagulation and fibrinolytic characteristics. We evaluated the affect of VLHL PAI-1 on haemostasis using the mouse model and showed that VLHL PAI-1 is an effective inhibitor of fibrin clot degradation. It reduced time of bleeding and total blood loss. CONCLUSION: VLHL PAI-1 may provide an important physiological mechanism to protect clots from premature dissolution in surgical and trauma settings.

[Cell phenotype determines PAI-1 antiproliferative effect - suppressed proliferation of the lung cancer but not prostate cancer cells]. / Fenotyp komórek warunkuje antyproliferacyjne dzialanie PAI-1 -hamujacy wplyw na aktywnosc proliferacyjna komórek raka pluca.

INTRODUCTION: Plasminogen inhibitor activator type 1 (PAI-1) is an important regulator of tumor growth and metastasis formation acting directly via specific urokinase complexing or indirectly due to its affinity to vitronectin. We have shown previously that PAI-1 modifies angiogenic activity of endothelial cells in a dose-dependent manner but also in close relationship to the cell phenotype. Present study aimed on evaluating the PAI-1 effect on the proliferative activity of lung cancer cells (A549), prostate cancer cells (DU145) as well as endothelial cells (HUVEC). RESULTS: Mutated PAI-1 (1, 10, 100 microg/mL) characterized by the prolonged antifibrinolytic activity (T1/2 approximately 7000 h) inhibited proliferation of lung cancer A549 cells in a dose-dependent (p < 0.001) and time-dependent (p < 0.001) manner. No significant effect on the DU145 prostate cancer cells has been observed except of the 72 h cultures with highest PAI-1 concentration (100 microg/ml) (p < 0.001). Proliferative activity of endothelial cells (HUVEC) was affected by 100 microg/ml PAI-1 only, and independent of the culture period (24, 48 and 72 h, p < 0.001). CONCLUSION: Plasminogen inhibitor activator type 1 modulates cell proliferation via antifibrynolitic mechanizm time- and dose-dependently, however final outcome is strongly affected by the cell phenotype.

VLHL plasminogen activator inhibitor spontaneously reactivates from the latent to active form.

Wild-type plasminogen activator inhibitor type 1 (PAI-1) is a fast-acting uPA and tPA inhibitor with half-life of 1-2 h. Recombinant PAI-1 with two mutations, Q197C and G355C, shows a very long half-life (VLHL). An introduced disulfide bridge holds together two central, parallel strands of beta-sheet A, preventing their separation to incorporate residues P4-P14 during the serpin's transition into latency. An active PAI-1 is usually described as a single structure with the reactive center loop (RCL) with P1-P1' (R369-M370) extended far from the bulk of the serpin's body. We have found that VLHL PAI-1 exists in several active forms that travel with different electrophoretic mobilities. Under aerobic conditions, two distinct active forms are observed. Upon reduction of cysteines, the VLHL mutant converts into the latent form, which spontaneously reactivates into a fully or partially active serpin, with yet another mobility. Utilizing electrophoresis, zymography (to check PAI-1 activity toward uPA) and theoretical calculations for molecular modeling, we have characterized active 1, 2, 3 and latent conformers of VLHL PAI-1 and their behaviors at normal and elevated temperatures, and in normal or reducing environments. VLHL PAI-1 activity is not affected, and the molecules do not polymerize unless reduced and/or heated. VLHL PAI-1 associates into dimers and bigger oligomers when -SH groups become available for oxidation and formation of intra- or intermolecular -S-S- bridges between conformers of different shapes and activities. We postulate that the active structures differ in RCL conformation and their position in relation to the gate region and the rest of the molecule.

Do human lipoxygenases have a PDZ regulatory domain?

Human lipoxygenases and products of their catalytic reaction have a well established connection to many human diseases. Despite their importance in inflammation, cancer, cardiorenal and other ailments the drug development is impaired by the lack of structural details to understand their intricate specificity and function in molecular and cellular signaling. The major effort so far has been directed towards understanding the determinants of their specificity and inhibition of their active site with the iron cofactor. Their structure is believed to consist of only two domains: one regulatory - a beta-sandwich, important for membrane binding, and one, mostly helical, catalytic domain. Although recently published cohort studies on single nucleotide polymorphism and occurrence of diseases, SAXS analysis and new biochemical data throw new light on lipoxygenase suggesting symbiosis of regulatory functions with an allosteric mechanism and more flexible structure than anticipated. The goal of this brief review is to direct an attention to the structural features of an anticipated topology and stimulate discussion/research to prove or disapprove our hypothesis that lipoxygenases may possess about approximately 110 amino acids PDZ-like fragments of functional importance. If they do have a second regulatory domain, it might help to explain their association with other molecules, role in signaling pathways and present a new avenue to explore the regulation of their behavior, and thus intervention in the course of diseases.

Proteolysis is the most fundamental property of malignancy and its inhibition may be used therapeutically (Review).

The mortality rates of cancer patients decreased by ~1.5% per year between 2001 and 2015, although the decrease depends on patient sex, ethnic group and type of malignancy. Cancer remains a significant global health problem, requiring a search for novel treatments. The most common property of malignant tumors is their capacity to invade adjacent tissue and to metastasize, and this cancer aggressiveness is contingent on overexpression of proteolytic enzymes. The components of the plasminogen activation system (PAS) and the metalloproteinase family [mainly matrix metalloproteinases (MMPs)] are overexpressed in malignant tumors, driving the local invasion, metastasis and angiogenesis. This is the case for numerous types of cancer, such as breast, colon, prostate and oral carcinoma, among others. Present chemotherapeutics agents typically attack all dividing cells; however, for future therapeutic agents to be clinically successful, they need to be highly selective for a specific protein(s) and act on the cancerous tissues without adverse systemic effects. Inhibition of proteolysis in cancerous tissue has the ability to attenuate tumor invasion, angiogenesis and migration. For that purpose, inhibiting both PAS and MMPs may be another approach, since the two groups of enzymes are overexpressed in cancer. In the present review, the roles and new findings on PAS and MMP families in cancer formation, growth and possible treatments are discussed.

PAI-1 induces cell detachment, downregulates nucleophosmin (B23) and fortilin (TCTP) in LnCAP prostate cancer cells.

Plasminogen activator inhibitor (PAI-1) is an anticancer agent that inhibits plasmin driven proteolysis, limiting angiogenesis and metastasis. In low concentrations it could induce cancer cell motility by interacting with urokinase (uPA), its receptor (uPAR), vitronectin and integrins. Active PAI-1 binds to uPA forming a complex with uPAR, while the latent form of PAI-1 does not. PAI-1 is found in both forms in the circulation. It is not clear which form acts as an anticancer agent and how it interacts with malignant cells. To investigate how these forms reduce angiogenesis or metastasis, we have created PAI-1 cysteine mutants in the active conformation (VLHL PAI-1) with an extended half-life that reaches approximately 700 h and its R369A mutant, which has an active conformation but cannot bind to uPA (VLHLNS PAI-1). Both VLHL PAI-1s convert into the latent form when treated with a reducing agent (DTT) that breaks disulfide bridges. Unexpectedly, during routine investigation of LnCAP cell proliferation, we have found that cells detach from the culture vessels regardless of PAI-1 conformation or activity. Further investigation showed that treatment of cancer cells with VLHL PAI-1 downregulated nucleophosmin, while all forms of PAI-1 downregulated fortilin. These two proteins are implicated in important cellular processes (cell growth, cell cycle, malignant transformation). This suggests that PAI-1, in addition to its well-known anticancer properties, plays an important role in cell signaling. We hope that by exploring PAI-1's structure and function we might be able to understand and separate the different effects of PAI-1 on cancer cells and develop more effective therapeutic strategies in cancer treatment.

Highly stable plasminogen activator inhibitor type one (VLHL PAI-1) protects fibrin clots from tissue plasminogen activator-mediated fibrinolysis.

Plasminogen activator inhibitor-1 (PAI-1) is the major specific inhibitor of tissue-type plasminogen activator (tPA) which mediates fibrin clot lysis through activation of plasminogen. Wild-type-PAI-1 (wPAI-1) is rapidly converted to the latent form (half-life of approximately 2 h) and loses its ability to inhibit tPA. We developed a very long half-life PAI-1 (VLHL PAI-1), a recombinant protein with a half-life >700 h compared with wPAI-1. In this study, VLHL PAI-1 was assessed for its ability to inhibit clot lysis in vitro. Clot formation was initiated in normal plasma supplemented with tPA by the addition of either tissue factor or human recombinant FVIIa. Clot lysis time, monitored turbidimetrically in a microtiter plate reader, was determined at various concentrations of wPAI-1 and VLHL PAI-1. Both wPAI-1 and VLHL PAI-1 caused a significant increase in clot lysis time, although the latter was somewhat less effective at lower concentrations. The VLHL PAI-1, but not wPAI-1, maintained its anti-fibrinolytic activity after preincubation overnight at 37 degrees. These studies demonstrate that VLHL PAI-1 is an effective inhibitor of fibrin clot degradation. Due to the high stability of VLHL PAI-1 compared with wPAI-1, this novel inhibitor of tPA-mediated fibrinolysis may have therapeutic applications for treating surgical and trauma patients when used directly or in conjunction with the procoagulant recombinant FVIIa.

Synthetic curcuminoids modulate the arachidonic acid metabolism of human platelet 12-lipoxygenase and reduce sprout formation of human endothelial cells.

Platelet 12-lipoxygenase (P-12-LOX) is overexpressed in different types of cancers, including prostate cancer, and the level of expression is correlated with the grade of this cancer. Arachidonic acid is metabolized by 12-LOX to 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], and this biologically active metabolite is involved in prostate cancer progression by modulating cell proliferation in multiple cancer-related pathways inducing angiogenesis and metastasis. Thus, inhibition of P-12-LOX can reduce these two processes. Several lipoxygenase inhibitors are known, including plant and mammalian lipoxygenases, but only a few of them are known inhibitors of P-12-LOX. Curcumin is one of these lipoxygenase inhibitors. Using a homology model of the three-dimensional structure of human P-12-LOX, we did computational docking of synthetic curcuminoids (curcumin derivatives) to identify inhibitors superior to curcumin. Docking of the known inhibitors curcumin and NDGA to P-12-LOX was used to optimize the docking protocol for the system in study. Over 75% of the compounds of interest were successfully docked into the active site of P-12-LOX, many of them sharing similar binding modes. Curcuminoids that did not dock into the active site did not inhibit P-12-LOX. From a set of the curcuminoids that were successfully docked and selected for testing, two were found to inhibit human lipoxygenase better than curcumin. False-positive curcuminoids showed high LogP (theoretical) values, indicating poor water solubility, a possible reason for lack of inhibitory activity or/and nonrealistic binding. Additionally, the curcuminoids inhibiting P-12-LOX were tested for their ability to reduce sprout formation of endothelial cells (in vitro model of angiogenesis). We found that only curcuminoids inhibiting human P-12-LOX and the known inhibitor NDGA reduced sprout formation. Only limited inhibition of sprout formation at approximately IC(50) concentrations has been seen. At IC(50), a substantial amount of 12-HETE can be produced by lipoxygenase, providing a stimulus for angiogenic sprouting of endothelial cells. Increasing the concentration of lipoxygenase inhibitors above IC(50), thus decreasing the concentration of 12(S)-HETE produced, greatly reduced sprout formation for all inhibitors tested. This universal event for all tested lipoxygenase inhibitors suggests that the inhibition of sprout formation was most likely due to the inhibition of human P-12-LOX but not other cancer-related pathways.

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.

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.

Plasminogen activator inhibitor-1 is locked in active conformation and polymerizes upon binding ligands neutralizing its activity.

Plasminogen activator inhibitor-1 (PAI-1), a member of the serpin super-family, forms a covalent complex with its target proteinases, such as tissue and urokinase plasminogen activators. Thus, PAI-1 controls the physiological and pathological proteolysis. An abnormal expression of PAI-1 has been observed in different diseases, which can be treated by returning the proteolysis back to normal physiological levels. It has been reported that some PAI-1 inhibitors neutralize its activity by accelerating the conversion of PAI-1 into a latent form. We have found small organic chemicals that also neutralize PAI-1 activity, but by a different mechanism. Using the NBD fluorescent probe [N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] incorporated into the reactive center loop (RCL) of PAI-1, we measured the kinetics of conversion from an active to a latent form. Unexpectedly, we found that some inhibitors of PAI-1 arrest this serpin in its active form instead of increasing the speed of conversion. Using docking calculations, we located two possible binding sites for these chemicals. The sites are in proximity of the P1/P1' amino acids of the RCL of PAI-1. Binding in this area can inactivate PAI-1 and additionally create a steric obstacle on the RCL making insertion of this loop between the A3 and A5 strands more difficult; hence abolishing a necessary step in the conversion of this protein into the latent form. Additionally, PAI-1 inhibitors link the RCL of one PAI-1 molecule with the strand 3C and strand 4C or helix A and strand 1B regions of the other PAI-1 molecule aiding polymerization or stabilizing the junction of the two. The polymerization of PAI-1 reduces PAI-1 activity by encapsulating the critical RCL fragment inside the formed PAI-1/PAI-1 polymers.

Determining whether curcumin degradation/condensation is actually bioactivation (Review).

Curcumin has been shown to exert therapeutic or protective effects against a variety of diseases, such as cancer, pulmonary diseases, neurological, liver, metabolic, autoimmune, cardiovascular diseases and numerous other chronic ailments. Over 116 clinical studies on curcumin in humans were registered with the US National Institutes of Health in 2015. However, it is mystifying how curcumin can be so effective in the treatment of many diseases since it has very low water solubility and bioavailability. Furthermore, curcumin is not stable under various conditions; its degradation or condensation into different bioactive compounds may be responsible for its biological activities rather than curcumin itself. In this review, we provide evidence of curcumin degradation and condensation into different compounds which have or may have health benefits themselves. Literature reviews strongly suggest that these molecules contribute to the observed health benefits, rather than curcumin itself.

Clinical implications of the growth-suppressive effects of chlorhexidine at low and high concentrations on human gingival fibroblasts and changes in morphology.

Chlorhexidine (CHX) is considered the gold standard in the antiseptic treatment of the oral cavity, due to its high antibactericidal capability. With the use of CHX mouth-rinse formulations, the bacteriostatic effects are maintained by the adsorption and prolonged release of CHX from oral surfaces. It was believed that antiplaque formation ability and the lack of systemic toxicity of CHX render it an excellent antiseptic in post-surgical dental treatment. However, recent studies have demonstrated that CHX exerts cytotoxic effects on human periodontal tissues, such as gingival fibroblasts and other cells. It also reduces gingival fibroblast adhesion to fibronectin and prevents fibroblast attachment to root surfaces, thus interfering with periodontal regeneration. In this study, using human gingival fibroblasts (HGFs), we investigated effects of CHX on the growth, morphology and proliferation of HGFs. We found that a low concentration (0.002%) of CHX does not interfere with the proliferation and morphology of HGFs. However, a higher concentration (≥0.04%) of CHX inhibits cell proliferation and to a certain extent, affects cell morphology in a time-dependent manner. A decrease in the percentage of cells in the G0/G1 phase and the accumulation of cells in the S phase following treatment with CHX also occurred in a dose-dependent manner. We thus concluded that CHX only at the concentration of 0.002% does not interfere with HGF growth, that is so critical to wound healing. Thus, the application of CHX in the post-surgical antiseptic treatment of the oral cavity should be limited.

In vivo and in vitro effect of baicalein on human prostate cancer cells.

We investigated the in vitro effects of baicalein and baicalin on human umbilical vein endothelial cells (HUVECs) and on human prostate tumor cells (DU-145 and PC3) as well as the effect of orally administered baicalein on the growth of DU-145 cells after subcutaneous injection into SCID mice. In vitro effects of baicalein and baicalin treatment on human prostate cancer cell lines DU-145 and PC-3 were assessed by employing cell proliferation (MTS) assay, cytotoxicity (LIVE/DEAD) assay, and TUNEL assay. In vitro anti-proliferative and anti-angiogenic properties of baicalein and baicalin were studied on HUVECs by sprout assay. The effect of orally administered baicalein on tumor growth in SCID mice was studied in four groups (n=10) of animals injected subcutaneously with DU-145 cells and treated daily for 28 days. The control group received only vehicle (carboxymethylcellulose), whereas the other three groups received escalating doses of baicalein (10, 20, and 40 mg/kg per day). Baicalein and baicalin exhibit dose-dependent growth inhibitory effects on human prostate cancer cells and umbilical vein endothelial cells in vitro. Also, treatment by these two flavonoid compounds significantly decreased the average number and length of sprouts formed by the endothelial cell aggregates in a dose-dependent manner. In vivo, treatment of mice with baicalein demonstrated a statistically significant tumor volume reduction (p<0.01) when compared to the control. This is the first study demonstrating an in vivo growth inhibitory effect of orally administered baicalein on human prostate tumors in mice.

A novel form of the plasminogen activator inhibitor created by cysteine mutations extends its half-life: relevance to cancer and angiogenesis.

Proteolytic activity driven by urokinase (uPA) is commonly recognized as an important factor in metastasis and angiogenesis. The eradication of unwanted uPA activity expressed by cancer cells results in the inhibition of metastasis and angiogenesis. Development of novel and highly selective uPA inhibitors could, therefore, produce new treatments of cancer. The ultimate goal of this work is the identification of novel and selective inhibitors of uPA suitable for this purpose. We have chosen plasminogen activator inhibitor(s) type 1 (PAI-1), which selectively inhibits the urokinase plasminogen activator (uPA). However, PAI-1 is not a stable molecule and converts itself into the latent form with a half-life in the range of t1/2 = 1-2 h. This conversion is associated with a partial insertion of the reactive loop (P4-P10') into the PAI-1 molecule. In such a conformation, P1-P1' and other sites are not accessible for reaction with uPA. To conquer this hurdle, we have produced several PAI-1 mutants by replacing chosen amino acids with cysteine in the hope of creating disulfide bridges, which could make this insertion more difficult. On the basis of the known structure of active PAI-1, we have identified amino acids that can be substituted with a cysteine residue to produce disulfide bridges linking the top and bottom parts of strands A3 and A5 as well as sites within the helix-D region. We created a total of seven cysteine mutants via point mutation (two to six point mutations), generating possible sites for disulfide bridge formation at the top and bottom parts of A3 and A5, within the helix-D region, or by a combination thereof. Desired mutations were introduced by PCR using appropriate primers. The mutant forms of PAI-1 containing the chitin-binding intein tag were then purified using affinity chromatography wherein the intein tag is cleaved, leaving mutant PAI-1 protein. Cys mutations resulted in proteins with extended half-life of PAI-1 from 2 to >700 h depending on the mutant. Novel PAI-1 were fully functional against uPA and showed activity in the in vitro model of angiogenesis, e.g., in the inhibition of sprout formation. Such prolonged serpin activity, which is therapeutically desired in cancer treatment and Cys-mutated PAI-1, could launch a new class of novel anticancer agents.