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Flavonoids are a group of polyphenolic compounds with different chemical structure and properties. These compounds may scavenge free radical species and other oxidants. In previous studies, we have observed that some polyphenols are able to cross the erythrocyte’smembrane and this process is influenced by the plasmatic albumin; in fact quercetin intracellular concentrations is albumin dose depending. The influence of flavonoids on catalase activity has been reported in some papers but the results are contradictory. Some authors have found an increase of catalase activity in cell in the presence of flavonoids. Others have observed any effect or even a decrease of catalase activity.The present work is based on a study of Krych’s, in which the influence of flavonoids on catalase in model system has been evaluated. Aim of this work was to study the role of red body cells in the antioxidant balance. The primary goal was to evaluate the antioxidant status of no-smokers and ever-smokers healthy subjects by the determination of the plasma antioxidant capacity and of the catalase activity of erythrocyte and then to evaluate if flavonoids (quercetin and luteolin) are able to modify the enzyme activity. This is a pilot study. Nine healthy subjects, aged 24-55 years were recruited. None of the subjects had any pathologies at the time of sampling. We assayed the CAT activity in erythrocytes isolated from whole blood of the subjects by the colorimetric assay and the plasma antioxidant capacity using the spectrophotometric method known crocin bleaching assay. The catalase activity was performed in human erythrocytes (control) and after the incubation of them with the flavonoids (quercetin and luteolin). The flavonoids were efficiently taken up by human erythrocytes in dose-depending manner. There was no significant difference in the percent accumulation of both molecules (quercetin and luteolin) inside the erythrocytes when incubated at the same concentration of 50μM. In physiologic condition the catalase activity varies from 28.6 mU/g protein to 40.6 mU/g protein. Data have shown that CAT activity of erythrocytes was significantly lower in ever smokers than in nosmokers. It was also found that Quercetin at the concentration of 100μM is able to increase the catalase levels in ever-smokers up to the normal values observed in no smokers. The study on the luteolin has not produced the same effects. In fact, Luteolin is able to reduce the CAT levels in no-smokers subject according with the data from Krych. The different actions of compounds on catalase can been explained as consequence of flavonoid interaction with enzymatic protein. The inhibiting action of the luteolin can be a consequence of a conformational change which occurs upon the flavonoid binding to catalase. This interaction changes the geometry of the substrate channel and thus inhibits the reaction of H2O2 with the heme center. Plasma antioxidant capacity was lower in no smokers than in smokers. An inverse correlation has been found between age and plasma antioxidant capacity. From the results of this study affirms that an oxidative stress condition is present in ever-smokers respect to non-smokers, but the quercetin is able to restore the erythrocyte oxidative stress condition of ever-smokers back to the normality. Further studies are necessary in the future to better investigate the role of luteolin on the catalase activity of human erythrocytes.

Background: The effects of the bisphosphonate derivative zoledronic acid (ZA) on the > circulating levels of matrix metalloproteinase-2 (MMP-2), matrix metallo-proteinases-9 > (MMP-9), cathepsin B (Cath B) and urokinase-type plasminogen activator (uPA) in > patients with bone metastasis (BMTS) and the possible correlation with the symptomatic > response induced by this drug in these patients were evaluated. Patients and Methods: > Proteinase levels were determined by enzyme-linked immunosorbent assay (ELISA) in the > plasma of 30 patients with painful bone metastases from breast or prostate cancer > undergoing multiple treatment with ZA (4 mg i.v., every 4 weeks). Healthy subjects > (HS) of both genders (12 female and 30 male) served as the control group. The > symptomatic response to ZA was assessed by the visual analog scale score (VAS). > Results: The median MMP-2 and MMP-9 pretreatment levels were more elevated in BMTS as > compared to HS (p¡Ü0.0001). Conversely, uPA levels were lower in BMTS p=0.0033; no > significant difference was observed for Cath B. ZA administration was associated with > a symptomatic response (VAS score¡Ü4) in 25/30 patients (83.3%) (p phenomenon paralleled a decrease of Cath B and MMP-2 plasma concentrations from > baseline values on week 12 (p=0.05). A similar trend, although not statistically > significant, was also noted for MMP-9 and uPA. However, no direct relationship was > observed between the analgesic effect induced by ZA and changes in the circulating > levels of these enzymes. Conclusion: These data show that ZA administration may > provide relief from bone pain in patients with diffuse skeletal metastases and confirm > a possible implication of cysteine proteinases and matrix metalloproteinases in bone > metastasis formation, but not in the pathogenesis of metastatic bone pain.

We have selected an etoposide-resistant variant (CCRF-CEM/VP-16) of the human T-lymphoblastic CCRF-CEM leukemia for study. Resistance to the topoisomerase II (topo II) inhibitor was about 11-fold and stable. Other data revealed that the new cell line had acquired an atypical, non-P-glycoprotein overexpressing multidrug resistant (MDR) phenotype with cross-resistance to other topo II inhibitors (amsacrine, doxorubicin, and mitoxantrone) and to glucocorticoids, but not to novobiocin, ICRF-187, vincristine or cisplatin. In a first instance, we assumed that altered drug-topo II interactions, based on quantitative and/or qualitative modifications of the enzyme, are a cause of resistance in the cell line. We tried to modify the drug sensitivity of the cells by means of various agents and cytokines. Positive results were obtained with verapamil and, to a lesser extent, cyclosporin A, but they were not specific for the drug resistant variant and occurred in the parental CCRF-CEM as well. Other attempts with buthionine sulfoximine, novobiocin, pentoxifylline, interleukin-1, interferon-alpha, retinoic acid, TNF-alpha, bryostatin 1 or phorbol myristate acetate were substantially unsuccessful, thus confirming the difficulty of pharmacologically overcoming atypical MDR. More encouragingly, however, CCRF-CEM/VP-16 cells exhibited hypersensitivity to other agents, including actinomycin D and taxol.

We have studied the interaction of glutathione-depleting concentrations of buthionine sulfoximine (BSO) with the anti-proliferative activity of doxorubicin (DXR) in three tumor lines, the mouse B16 melanoma. Friend erythroleukemia and the human K562 leukemia, both as DXR-sensitive and-resistant (with typical multidrug resistance) variants. BSO significantly enhanced the DXR effects in the wild-type Friend and K562 leukemias, and especially in the drug-resistant subline of Friend leukemia. BSO did not modify DXR accumulation and retention in the latter clone. Moreover, neither BSO nor verapamil used alone completely reversed the resistance to DXR of this cell line; their combination was more efficient and increased its drug sensitivity to a level closer to that of the parental counterpart. These results seem to indicate that the status of glutathione and of the enzymes related to it contributes to the resistance of Friend leukemia to DXR. An interesting additional finding was that BSO significantly synergizes with the antiproliferative effects of vincristine in the drug-sensitive variants of Friend and K562 leukemias.

There is emerging clinical evidence that amsacrine (m-AMSA) administration may be associated with cardiotoxic effects such as severe, even fatal, ventricular arrhythmias and impairment of the inotropic performance of the heart. Information on the cardiac effects of m-AMSA in animals is scanty. Studies on mice, dogs, and monkeys have not evidenced the cardiotoxicity of the compound. The data presented in this paper show that m-AMSA causes acute ECG alterations in normal rabbits and a dose-related negative inotropic effect on the isolated rabbit heart, suggesting that this species may be a useful model for the study of the cardiac actions of this antiblastic.

Growing evidence indicates that lysosomal Cathepsins D (CD), B (CB) and L (CL) may promote carcinogenesis and tumor progression. Therefore, we evaluated their potential value as biochemical parameters of malignant progression in patients with benign diseases which may undergo malignant transformation, such as liver cirrhosis (LC) and chronic pancreatitis (CHP) as well as in hepatocellular carcinoma (HCC) and pancreatic cancer (DPC). CD, CB and CL serum levels were determined by immunoenzymatic assays in LC, CHP, HCC or DPC patients and correlated with a number of biochemical and clinical parameters of these diseases. CD serum levels were increased in LC, CHP and HCC, but not in the DPC group as compared to normal subjects (NS) (P < 0.01). Interestingly, higher levels of this enzyme were observed in LC patients compared to HCC patients (P < 0.01). CB serum concentrations were increased in all patient groups (P < 0.01). However no difference was evidenced between benign and malignant diseases. CL serum levels were significantly increased only in DPC as compared to NS (P < 0.01) or CHP patients (P < 0.02) and in HCC as compared to NS (P < 0.01). The evaluation of CD, CB and CL serum pattern in LC, CHP, HCC and DPC patients may be useful as additional biochemical parameters in the differential diagnosis and therapeutic monitoring of these diseases. Prospective clinical investigations to assess the potential value of these enzymes as biochemical markers of malignant progression of LC or CHP are warranted by the present data.

This study was directed to assess the clinical impact of the circulating cathepsin L, cystatin C, activin A, and follistatin in breast cancer patients. The serum concentrations of these molecules were determined by immunoenzymatic assays, and their association with some clinico-pathological parameters of breast cancer progression was evaluated. Our results identified cystatin C and activin A as predictive markers for the presence of breast cancer and bone metastasis, respectively. Therefore, these proteins may have a clinical role as circulating biomarkers in the diagnosis and therapeutic monitoring of breast cancer patients.

Cathepsin L is a ubiquitous lysosomal cysteine endopeptidase that is mainly involved in the metabolic turnover of intracellular proteins. However, it is now well established that this enzyme may also be implicated in the regulation of other important biological processes includ- ing bone resorption. Therefore, altered expression levels of Cathepsin L may result in disturbances of bone homeo- stasis and, eventually, in the onset of pathological condi- tions associated with altered bone turnover. These observations support the concept that Cathepsin L may be regarded as an additional target for the development of novel therapeutic options for the treatment of patients with bone diseases. This review provides insight into the most recent advances in understanding the role of Cathepsin L in normal and pathological bone remodeling and discusses the potential clinical and therapeutic applications related to these findings.

Context: There is increasing evidence that Vitamin D (Vit D) and its metabolites, besides their well-known calcium-related functions, may also exert antiproliferative, pro-differentiating, and immune modulatory effects on tumor cells in vitro and may also delay tumor growth in vivo. Objective: The aim of this review is to provide fresh insight into the most recent advances on the role of Vit D and its analogues as chemopreventive drugs in cancer therapy. Methods: A systematic review of experimental and clinical studies on Vit D and cancer was undertaken by using the major electronic health database including ISI Web of Science, Medline, PubMed, Scopus and Google Scholar. Results and conclusion: Experimental and clinical observations suggest that Vit D and its analogues may be effective in preventing the malignant transformation and/or the progression of various types of human tumors including breast cancer, prostate cancer, colorectal cancer, and some hematological malignances. These findings suggest the possibility of the clinical use of these molecules as novel potential chemopreventive and anticancer agents

The oxidative stress results from a change in the physiological balancebetween oxidant and antioxidant species. This condition induces an chemical change in the redox state of cells.. The function of t-PMET is to cooperate with intracellular redox pairs, like piruvate and lactate, to maintain thecytoplasmatic NAD+/NADH balance. The t-PMET is correlated with the modulation of internal pH and redox homeostasis, as it is able to activate proton release. Thus, t-PMET causes external and internal pH modification, as well as development of an inside negative membrane potential. The aim of this work was evaluate the erythrocytes redox status in a group of health volunteers and then to study whether some of flavonoids, enclosed in sub-class of flavonols (Quercetin and Kaempferol), are able to modify the erythrocytes redox homeostasis.Our attention has been focused on red blood cells (RBC) because a close link between t-PMET and metabolic status of erythrocytes has been reported. The RBCs act as antioxidants to themselves and, inaddition, their mobility carries their antioxidant capabilities to all the plasma accessible parts of the body.The subjects that participating at this work attested no supplement intake or some other substance that could interfere with our tests. Human venous blood from twenty healthy volunteers of both sexes between the ages of 25-50 years were obtained by venipuncture in heparin after an over-night fast and centrifuged.The antioxidant capacity of plasma was analyzed by crocin bleaching assay and FRAP. On the other hand, the reducing activity in erythrocytes that represents the body redox state of the last 120 days was evaluated by FRAP method. After centrifugation at 3000 rpm for 10 minutes at 4°C, plasma was separate from red blood cells. The resultant plasma was transferred to microcentrifuge tubes and used at least in part for ferric-reducing activity power (FRAP) and for Crocin Bleaching Assay (CBA) and the others aliquots was stored -80°C before to use for further analysis. Red blood cells, after removal of buffy coat and upper 15% of the packed red blood cells, were washed two times with cold PBS. A stock solution (20mM) of each flavonoid was prepared in dimethyl sulfoxide and then diluted 1:2 with PBS. Packed RBC (10%v/v) were incubatedin PBS containing 5mM glucose at 37°C for 10. minutes with a 50 μM concentration of each flavonoids. After this time the suspensions was centrifuged, the RBC were washed and then analyzed. The percentage of hemolysis was evaluated in the same sample by measuring the haemoglobin contents.The extent of lysis was not different than the controls and never higher than 0.5%.All the values confirm the data of literature about the antioxidant status of human being in physiological condition. All compounds were taken up by the erythrocytes and displaying significant FIC-reducing activity. Both the analysed compounds (Quercetin and Kaempferol) are able to increase the reducing activity of the erythrocytes of 15% and 13% respectively respect to the value recorded for the control. On the other hand, only the quercetin was able to increment the activity of the tPMET system; not any significant difference has been recorded between the Kaempferol and the control group .This study shows that the flavonoids are able to form stable complexeswith the erythrocytes and to influence the intracellular redox homeostasis.Therefore, it could affirm that the polyphenols are able to increase the defence of erythrocytes against ROS. This work underlines that the RBC plays a pivotal role in the distribution and bioavailability of circulating polyphenols which contribute to the defence against injury induced by ROS in various clinical disorders.

Experiments were performed to assess the dose-dependent effects of quercetin, kaempferol, (+) catechin, and (-) epicatechin on superoxide radical production through the modulation of manganese superoxide dismutase and xanthine oxidase activities. The experiments were carried out at flavanoid concentrations ranging from 1 µM to 100 µM. This investigation highlighted that flavonols induced opposite effects on superoxide radical production at different doses, i.e. pro-oxidant at the highest concentration (100 µM) and anti-oxidant at the lowest concentration (1 µM). Similar behaviors were observed for xanthine oxidase with flavan-3ols. The diastereoisomer (the catechin) acted as a stronger radical scavenger than the epicatechin. However, flavan-3ols were less pro-oxidant than flavonols: in fact, the addition of the superoxide dismutase enzyme was able to cancel the flavan-3ols' pro-oxidant effect. This study also shows that the absence of the 4-carbonyl group conjugated with the 2-3 double bonds in the heterocyclic ring cancelled the pro-oxidant effect of flavan-3ols. The opposite dose-dependent effects of flavonols suggest that they may be used as either a pro-oxidant or antioxidant.

Follistatin is a single-chain glycosylated protein whose primary function consists in binding and neutralizing some members of the transforming growth factor-β superfamily such as activin and bone morphogenic proteins. Emerging evidence indicates that this molecule may also play a role in the malignant progression of several human tumors including prostate cancer. In particular, recent findings suggest that, in this tumor, follistatin may also contribute to the formation of bone metastasis through multiple mechanisms, some of which are not related to its specific activin or bone morphogenic proteins' inhibitory activity. This review provides insight into the most recent advances in understanding the role of follistatin in the prostate cancer progression and discusses the clinical and therapeutic implications related to these findings.

Several studies report that the plasma antioxidant capacity (PAC) increases after a single ingestion of red wine. However, data on the different behaviour of PAC after food intake in subjects of the same sex are still lacking. On the basis of these observations some investigations were undertaken in order to evaluate the effects of red wine intake on PAC in a homogeneous groups of healthy female volunteers (n=18). Additionally, the possible correlation between increase of PAC values and increase in uric acid levels was also assessed. PAC and uric acid concentrations were determined before wine intake and 50, 120 and 240 minutes thereafter. The results obtained following these studied highlighted two different patterns of variation of plasma AC values after red wine intake in women. Group “A” exhibits a significant increase in PAC at 120 minutes after wine consumption while group “B” showed a peak level of AC 50 minutes after wine intake. However, no significant correlation was highlighted between increased levels of uric acid and PAC. These results provide a strong argument for the hypothesis that sampling procedures may be one of the confounding factor in studies on the plasma antioxidant status after food or beverage consumption. These preliminary observations indicate that sex-based selection of volunteers should be considered in further investigations.

Cathepsin L is a lysosomal cysteine proteinase primarily devoted to the metabolic turnover of intracellular proteins. However, accumulating evidence suggests that this endopeptidase might also be implicated in the regulation of other important biological functions, including bone resorption in normal and pathological conditions. These findings support the concept that cathepsin L, in concert with other proteolytic enzymes involved in bone remodeling processes, could contribute to facilitate bone metastasis formation. In support of this hypothesis, recent studies indicate that cathepsin L can foster this process by triggering multiple mechanisms which, in part, differ from those of the major cysteine proteinase of osteoclasts, namely cathepsin K. Therefore, cathepsin L can be regarded as an additional target in the treatment of patients with metastatic bone disease. This review discusses the clinical and therapeutic implications related to these findings.

The clinical significance of circulating fol- listatin (FLST), an inhibitor of the multifunctional cytokine activin A (Act A), was investigated in patients with prostate cancer (PCa). The serum concentrations of this molecule were determined by an enzyme-linked immunosorbent assay (ELISA) in PCa patients with (M?) or without (M0) bone metastases, in patients with benign prostate hyperplasia (BPH) and in healthy sub- jects (HS). The effectiveness of FLST in detecting PCa patients with skeletal metastases was determined by the receiver operating characteristic (ROC) curve analysis. Serum FLST was significantly higher in PCa patients than in BPH patients (P = 0.001) or HS (P = 0.011). Conversely, in BPH patients, FLST levels resulted lower than in HS (P = 0.025). In cancer patients the serum concentrations of FLST significantly correlated with the presence of bone metastases (P = 0.0005) or increased PSA levels (P = 0.04). Interestingly, significant differ- ences in the ratio between FLST and Act A serum concentrations (FLST/Act A) were observed between HS and BPH patients (P = 0.001) or PCa patients (P = 0.0005). Finally, ROC curve analysis, highlighted a sound diagnostic performance of FLST in detecting patients (P = 0.0001). However, the diagnostic effectiveness of FLST did not result significantly supe- rior to that of Act A or PSA. These findings suggest that FLST may be regarded as a potential, molecular target in the treatment of metastatic bone disease while its clinical role as soluble marker in the clinical management of PCa patients with bone metastases needs to be better defined.

The clinical significance of serum cathepsin K and cystatin C was assessed in patients with breast cancer (BCa) or prostate cancer (PCa) with confined disease (M0) or bone metastasis (BM). Cathepsin K and cystatin C circulating levels were determined by ELISAs in 63 cancer patients, in 35 patients with nonmalignant diseases and in 42 healthy blood donors (control group). In BCa patients, cathepsin K serum levels were significantly lower than in sex matched control group (HS; p = 0.0008) or in patients with primary osteoporosis (OP; p = 0.0009). On the contrary, cystatin C levels were significantly higher in BCa patients than in HS ( p = 0.0001) or OP ( p = 0.017). In PCa patients, cathepsin K concentrations did not significantly differ from those measured in sex matched HS or in patients with benign prostatic hyperplasia (BPH). Conversely, cystatin C was more elevated in cancer patients than in controls ( p = 0.0001) or BPH patients ( p = 0.0078). Furthermore, in PCa patients, a positive correlation was observed between cystatin C and cathepsin K ( r S = 0.34; p = 0.047). No further relationship was highlighted between these molecules and the clinicobiological parameters of BCa or PCa progression including the number of bone lesions. Moreover, ROC curve analysis showed a poor diagnostic performance of cathepsin K and cystatin C in the detection of BM patients. Interestingly, the administration of zoledronic acid (ZA), a bisphosphonate derivative endowed with a potent antiosteoclastic activity, induced in BM patients a marked increase of cathepsin K and cystatin C serum levels compared to baseline values. However, this phenomenon was statistically significant only in the PCa group. In conclusion Cystatin C and cathepsin K may be regarded as possible markers to monitor the therapeutic response to bisphosphonate treatments. Nevertheless, their clinical value as specific gauges of skeletal metastasis remains questionable.

Recent studies have highlighted that Activin A, a member of the transforming growth factor-beta (TGF-beta) superfamily, may be involved in the regulation of osteoblastic activity and in osteoclast differentiation. Therefore, we have investigated the clinical significance of its circulating levels in patients with bone metastasis. Activin A serum concentrations were determined, by a commercially available enzyme-linked immunosorbent assay kit, in 72 patients with breast cancer (BC) or prostatic cancer (PC) with (BM+) or without (BM-) bone metastases, in 15 female patients with age-related osteoporosis (OP), in 20 patients with benign prostatic hypertrophy (BPH) and in 48 registered healthy blood donors (HS) of both sex (25 female and 23 male). Activin A serum concentrations were significantly increased in BC or PC patients as compared to OP (P < 0.0001) or BPH (P = 0.045), respectively, or to sex matched HS (P < 0.0001). Additionally, these levels resulted more elevated in PC patients as compared to BC patients (P = 0.032). Interestingly, Activin A was significantly higher in BM+ patients than in BM- patients (BC, P = 0.047; PC, P = 0.016). In BC patients, a significant correlation was observed only between Activin A and number of bone metastases (P = 0.0065) while, in PC patients, Activin A levels were strongly correlated with the Gleason score (P = 0.011) or PSA levels (P = 0.0001) and, to a lessen extent, with the number of bone metastases (P = 0.056). Receiver operating characteristic curve (ROC) analysis showed a fair diagnostic accuracy of Activin A to discriminate between BM+ and BM- patients (BC: AUC = 0.71 +/- 0.09, P = 0.03; PC: AUC = 0.73 +/- 0.081, P = 0.005). These findings indicate that Activin A may be implicated in the pathogenesis of bone metastasis. Therefore, this cytokine may be considered a novel potential target for a more selective therapeutic approach in the treatment of skeletal metastasis and may be also useful as additional biochemical marker of metastatic bone disease.

Cathepsin D is a lysosomal acid proteinase which is involved in the malignant progression of breast cancer and other gynecological tumors. Clinical investigations have shown that in breast cancer patients cathepsin D overexpression was significantly correlated with a shorter free-time disease and overall survival, whereas in patients with ovarian or endometrial cancer this phenomenon was associated with tumor aggressiveness and a degree of chemoresistance to various antitumor drugs such as anthracyclines, cis-platinum and vinca alkaloids. Therefore, a lot of research has been undertaken to evaluate the role and the prognostic value of cathepsin D also in other solid neoplasms. However, conflicting results have been generated from these studies. The discrepancies in these results may, in part, be explained with the different methodological approaches used in order to determine the levels of expression of the enzyme in tumor tissues and body fluids. Further investigations using well-standardized techniques may better define the clinical significance of cathepsin D expression in solid tumors. Nevertheless, evidence emerging from these studies indicates that this proteinase seems to facilitate early phases of tumor progression such as cell proliferation and local dissemination. These findings support the concept that cathepsin D may be a useful marker for identifying patients with highly malignant tumor phenotypes who may need more aggressive clinical treatment; this enzyme may also be considered as a potential target for a novel therapeutic approach in the treatment of solid neoplasms.