Effects of oleuropein on tumor cell growth and bone remodelling: Potential clinical implications for the prevention and treatment of malignant bone diseases.

Oleuropein (Ole) is the main bioactive phenolic compound present in olive leaves, fruits and olive oil. This molecule has been shown to exert beneficial effects on several human pathological conditions. In particular, recent preclinical and observational studies have provided evidence that Ole exhibits chemo-preventive effects on different types of human tumors. Studies undertaken to elucidate the specific mechanisms underlying these effects have shown that this molecule may thwart several key steps of malignant progression, including tumor cell proliferation, survival, angiogenesis, invasion and metastasis, by modulating the expression and activity of several growth factors, cytokines, adhesion molecules and enzymes involved in these processes. Interestingly, experimental observations have highlighted the fact that most of these signalling molecules also appear to be actively involved in the homing and growth of disseminating cancer cells in bones and, ultimately, in the development of metastatic bone diseases. These findings, and the experimental and clinical data reporting the preventive activity of Ole on various pathological conditions associated with a bone loss, are indicative of a potential therapeutic role of this molecule in the prevention and treatment of cancer-related bone diseases. This paper provides a current overview regarding the molecular mechanisms and the experimental findings underpinning a possible clinical role of Ole in the prevention and development of cancer-related bone diseases.

On the role of cystatin C in cancer progression.

Cystatin C (Cyst C) is an endogenous inhibitor of lysosomal cysteine proteinases, which has been shown to play a role in several normal and pathological processes. Interestingly, a growing number of experimental and clinical studies suggest that this inhibitor also appears to be implicated in the malignant progression of various human tumors. However, the role of Cyst C in malignant diseases is still controversial as these studies have highlighted that this protein may function either as tumor suppressor or tumor promoter. The specific mechanisms underlying these opposing effects at present remain murky and are the subject of many current investigations. On the other hand, a complete knowledge of these mechanisms is of clinical interest in order to develop new, effective antitumor treatments based on the appropriate use of natural and/or synthetic cysteine proteinase inhibitors. This paper discusses the current findings regarding the role of Cyst C in cancer progression and the clinical implications emerging from these studies.

Clinical Impact of Cystatin C/Cathepsin L and Follistatin/Activin A Systems in Breast Cancer Progression: A Preliminary Report.

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.

Flavonols and flavan-3-ols as modulators of xanthine oxidase and manganese superoxide dismutase activity.

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 as potential therapeutic target in prostate cancer.

Follistatin is a single-chain glycosylated protein whose primary function consists in binding and neutralizing some members of the transforming growth factor-ß 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.

Anti-proliferative and pro-apoptotic activities of hydroxytyrosol on different tumour cells: the role of extracellular production of hydrogen peroxide.

PURPOSE: Several recently published data suggest that the anti-proliferative and pro-apoptotic properties of hydroxytyrosol [3,4-dihydroxyphenyl ethanol (3,4-DHPEA)] on HL60 cells may be mediated by the accumulation of hydrogen peroxide (H2O2) in the culture medium. The aim of this study was to clarify the role played by H2O2 in the chemopreventive activities of 3,4-DHPEA on breast (MDA and MCF-7), prostate (LNCap and PC3) and colon (SW480 and HCT116) cancer cell lines and to investigate the effects of cell culture medium components and the possible mechanisms at the basis of the H2O2-producing properties of 3,4-DHPEA. METHODS: The proliferation was measured by the MTT assay and the apoptosis by both fluorescence microscopy and flow cytometry. The concentration of H2O2 in the culture medium was measured by the ferrous ion oxidation-xylenol orange method. RESULTS: It was found that the H2O2-inducing ability of 3,4-DHPEA is completely prevented by pyruvate and that the exposure of cells to conditions not supporting the H2O2 accumulation (addition of either catalase or pyruvate to the culture medium) inhibited the anti-proliferative effect of 3,4-DHPEA. Accordingly, the sensitivity of the different cell lines to the anti-proliferative effect of 3,4-DHPEA was inversely correlated with their ability to remove H2O2 from the culture medium. With regard to the mechanism by which 3,4-DHPEA causes the H2O2 accumulation, it was found that superoxide dismutase increased the H2O2 production while tyrosinase, slightly acidic pH (6,8) and absence of oxygen (O2) completely prevented this activity. In addition, different transition metal-chelating compounds did not modify the H2O2-producing activity of 3,4-DHPEA. CONCLUSIONS: The pro-oxidant activity of 3,4-DHPEA deeply influences its 'in vitro' chemopreventive activities. The main initiation step in the H2O2-producing activity is the auto-oxidation of 3,4-DHPEA by O2 with the formation of the semiquinone, superoxide ions (O2(-)) and 2H(+).

Influence of cultivar and concentration of selected phenolic constituents on the in vitro chemiopreventive potential of olive oil extracts.

One of the main olive oil phenolic compounds, hydroxytyrosol (3,4-DHPEA), exerts in vitro chemopreventive activities (antiproliferative and pro-apoptotic) on tumor cells through the accumulation of H(2)O(2) in the culture medium. However, the phenol composition of virgin olive oil is complex, and 3,4-DHPEA is present at low concentrations when compared to other secoiridoids. In this study, the in vitro chemopreventive activities of complex virgin olive oil phenolic extracts (VOO-PE, derived from the four Italian cultivars Nocellara del Belice, Coratina, Ogliarola, and Taggiasca) were compared to each other and related to the amount of the single phenolic constituents. A great chemopreventive potential among the different VOO-PE was found following this order: Ogliarola > Coratina > Nocellara > Taggiasca. The antiproliferative and pro-apoptotic activities of VOO-PE were positively correlated to the secoiridoid content and negatively correlated to the concentration of both phenyl alcohols and lignans. All extracts induced H(2)O(2) accumulation in the culture medium, but this phenomenon was not responsible for their pro-apoptotic activity. When tested in a complex mixture, the olive oil phenols exerted a more potent chemopreventive effect compared to the isolated compounds, and this effect could be due either to a synergistic action of components or to any other unidentified extract constituent.

Serum follistatin in patients with prostate cancer metastatic to the bone.

The clinical significance of circulating follistatin (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 subjects (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 differences 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 M+ patients (P = 0.0001). However, the diagnostic effectiveness of FLST did not result significantly superior 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.

Cathepsin L in metastatic bone disease: therapeutic implications.

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.

Circulating cathepsin K and cystatin C in patients with cancer related bone disease: clinical and therapeutic implications.

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.

Activin A circulating levels in patients with bone metastasis from breast or prostate cancer.

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.

Effects of zoledronic acid on proteinase plasma levels in patients with bone metastases.

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 < or = 0.0001). 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 < or =4) in 25/30patients (83.3%) (p < 0.0001). This 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.

Cathepsin D expression levels in nongynecological solid tumors: clinical and therapeutic implications.

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 studied 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.