Maria Vittoria Sepporta

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

PurposeSeveral 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.MethodsThe 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.ResultsIt 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.ConclusionsThe 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.

Cathepsin L in metastatic bone disease: therapeutic implications

Abstract 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.

Pinoresinol Inhibits Proliferation and Induces Differentiation on Human HL60 Leukemia Cells

Pinoresinol (PIN), one of the simplest lignans, is the precursor of other dietary lignans that are present in whole-grain cereals, legumes, fruits, and other vegetables. Several experimental and epidemiological evidences suggest that lignans may prevent human cancer in different organs. In this study we investigated the chemopreventive properties of PIN on cell lines derived from different sites either expressing or not the functional tumor suppressor protein p53. It was found that PIN inhibited the proliferation of p53 wild type colon and prostate tumor cells (HCT116 and LNCaP) while in breast cells the inhibition of growth was observed only in p53 mutant cells (MDA-MB-231). A potent antiproliferative activity of PIN was also observed on p53 null cells HL60 (IC50% 8 μM), their multidrug resistant variant HL60R (IC50% 32 μM) and K562. On HL60 cells, PIN caused a block of cell cycle in the G0/G1 phase, induced a weak proapoptotic effect but it was a good trigger of differentiation (NBT reduction and CD11b expression). PIN caused an upregulation of the CDK inhibitor p21WAF1/Cip1 both at mRNA and protein levels so suggesting that this could be a mechanism by which PIN reduced proliferation and induced differentiation on HL60 cells.

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-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.

The hydroxytyrosol-dependent increase of TNF-α in LPS-activated human monocytes is mediated by PGE2 and adenylate cyclase activation

An accurate regulation of PGE2 and TNF-α production is an important event for a physiological inflammation process. We have recently reported that in LPS-activated human monocytes hydroxytyrosol, the main phenol present in extra virgin olive oil reduced both the COX-2 gene expression and PGE2 secretion while it increased the TNF-α accumulation in the culture medium. Here we have investigated whether these effects were related to each other, clarifying the possible mechanisms involved. We found that hydroxytyrosol (100 μM) increased the TNF-α mRNA level in LPS-activated human monocytes as evaluated by both RT-PCR and real time PCR (qPCR). Exogenous PGE2 reduced both TNF-α mRNA and TNF-α secretion (EIA assay) while the activation of adenylate cyclase by forskolin decreased only the TNF-α secretion but did not influence the TNF-α mRNA level. Acting similarly to non steroidal anti-inflammatory drugs (NSAIDs), the hydroxytyrosol could be used to develop innovative drugs for the control of inflammation and immune response. The decrease of TNF mediated by forskolin, moreover, could suggest that the pharmacological regulation of cAMP production may represent a strategy to control the side effects of NSAIDs.

Enhanced chemopreventive activity of hydroxytyrosol on HL60 and HL60R cells by chemical conversion into thio derivatives.

Thio derivatives of hydroxytyrosol containing thiol, thioacetate and disulfide functionalities were synthesized from natural hydroxytyrosol (3,4-DHPEA) via 3,4-dihydroxyphenethyl halides. These compounds, containing the combination of catechol moiety and divalent sulfur functions, were tested for the pro-apoptotic and anti-proliferative activities on both parental HL60 and multi-drug resistant HL60R cells. It was found that all synthesized compounds were more effective than 3,4-DHPEA in inducing apoptosis on HL60R cells, and that the hydroxytyrosol disulfide was the most active pro-apoptotic and anti-proliferative compound on both HL60 and HL60R cells. Different from 3,4-DHPEA, all thio derivatives of hydroxytyrosol induced apoptosis by a mechanism not involving the release of H(2)O(2) in the culture medium. The data on HL60R cells suggest that these compounds could be able to reverse the resistance toward the most common drugs in cancer therapy.

Preventive Activity of Olive Oil Phenolic Compounds on Alkene Epoxides Induced Oxidative DNA Damage on Human Peripheral Blood Mononuclear Cells

The aim of this study was to investigate the ability of epoxides of styrene (styrene-7,8-oxide; SO) and 1,3-butadiene (3,4-epoxy-1-butene; 1,2:3,4:-diepoxybutane) to cause oxidative stress and oxidative DNA damage on human peripheral blood mononuclear cells (PBMCs) and whether a complex mixture of olive oil phenols (OOPE) could prevent these effects. The DNA damage was measured by the single-cell gel electrophoresis (SCGE; comet assay). We found that the DNA damage induced by alkene epoxides could be prevented by N-acetyl-cysteine (10 mM) and catalase (100 U/ml). Alkene epoxides caused a significant (P < 0.05) increase of both peroxide concentration in extra- and intracellular environment and formamidopyrimidine DNA glycosylase (FPG)- and Endonuclease III (ENDO III)-sensitive sites in PBMCs, demonstrating the presence of oxidized bases. OOPE (1 μg of total phenols/ml) was able to prevent the alkene epoxide induced DNA damage both after 2 and 24 h of incubation. In addition, OOPE completely inhibited the SO-induced intracellular peroxide accumulation in PBMCs and prevented the oxidative DNA damage induced by SO, as evidenced by the disappearance of both FPG- and ENDO III-sensitive sites. This is the first study demonstrating the ability of OOPE to prevent the DNA damage induced by alkene epoxides providing additional information about the chemopreventive properties of olive oil.

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

ABSTRACT 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.