José Juan Gaforio

Olive oil and health: Summary of the II international conference on olive oil and health consensus report, Jaén and Córdoba (Spain) 2008

Olive oil (OO) is the most representative food of the traditional Mediterranean Diet (MedDiet). Increasing evidence suggests that monounsaturated fatty acids (MUFA) as a nutrient, OO as a food, and the MedDiet as a food pattern are associated with a decreased risk of cardiovascular disease, obesity, metabolic syndrome, type 2 diabetes and hypertension. A MedDiet rich in OO and OO per se has been shown to improve cardiovascular risk factors, such as lipid profiles, blood pressure, postprandial hyperlipidemia, endothelial dysfunction, oxidative stress, and antithrombotic profiles. Some of these beneficial effects can be attributed to the OO minor components. Therefore, the definition of the MedDiet should include OO. Phenolic compounds in OO have shown antioxidant and anti-inflammatory properties, prevent lipoperoxidation, induce favorable changes of lipid profile, improve endothelial function, and disclose antithrombotic properties. Observational studies from Mediterranean cohorts have suggested that dietary MUFA may be protective against age-related cognitive decline and Alzheimers disease. Recent studies consistently support the concept that the OO-rich MedDiet is compatible with healthier aging and increased longevity. In countries where the population adheres to the MedDiet, such as Spain, Greece and Italy, and OO is the principal source of fat, rates of cancer incidence are lower than in northern European countries. Experimental and human cellular studies have provided new evidence on the potential protective effect of OO on cancer. Furthermore, results of case-control and cohort studies suggest that MUFA intake including OO is associated with a reduction in cancer risk (mainly breast, colorectal and prostate cancers).

Antioxidant, Antiproliferative, and Pro-apoptotic Capacities of Pentacyclic Triterpenes Found in the Skin of Olives on MCF-7 Human Breast Cancer Cells and Their Effects on DNA Damage

This research aimed to investigate erythrodiol, uvaol, oleanolic acid, and maslinic acid scavenging capacities and their effects on cytotoxicity, cell proliferation, cell cycle, apoptosis, reactive oxygen species (ROS) level, and oxidative DNA damage on human MCF-7 breast cancer cell line. The results showed that erythrodiol, uvaol, and oleanolic acid have a significant cytotoxic effect and inhibit proliferation in a dose- and time-dependent manner. At 100 μM, erythrodiol growth inhibition occurred through apoptosis, with the observation of important ROS production and DNA damage, whereas uvaol and oleanolic acid growth inhibition involved cell cycle arrest. Moreover, although all tested triterpenes did not show free radical scavenging activity using ABTS and DPPH assays, they protected against oxidative DNA damage at the concentration 10 μM. Uvaol and oleanolic and maslinic acids, tested at 10 and 100 μM, also reduced intracellular ROS level and prevented H(2)O(2)-induced oxidative injury. Overall, the results suggest that tested triterpenes may have the potential to provide significant natural defense against human breast cancer.

Analysis of HLA expression in human tumor tissues.

Abstract. Cancer cells can be detected and destroyed by cytotoxic T lymphocytes in many experimental tumor systems, and – as has been well-documented – in some human tumors. In humans however, most diagnosed tumors are not eliminated by T cells but grow steadily, invading and metastasizing until the host is destroyed. Evidence is accumulating that progressive tumor growth occurs not because the immune system is defective or deteriorated, but because the cancer cell is capable of developing a variety of strategies to escape immune recognition. In addition, cancer cells acquire new biological properties to generate invasive capacity in order to migrate and colonize new tissues. Major histocompatibility complex (MHC) antigens are molecules that are specialized in communicating with the T cell receptor and natural killer (NK) cell ligands. With the former, they use the interaction with peptides derived from processed cellular and exogenous proteins to monitor self and non-self status. With the latter, they determine the degree of activation and killing capacity of NK cells by interacting with NK receptors. Any change in the MHC profile of tumor cells (including classical and nonclassical MHC molecules) may therefore have a profound influence on the immune recognition and immune rejection of cancer cells. We have reviewed the data from our laboratory and other groups, and have presented a standardized procedure for analyzing the MHC profile of human tumors with special emphasis on the quality and laboratory use of the material obtained from microdissected tumor samples. Appropriate tissue processing is of particular relevance, since it is not possible to obtain tumor cell lines from most patients. Oncologists require rapid information on the MHC profile of the tumor if gene therapy is envisaged to restore normal MHC class I gene expression.

MHC class I‐deficient metastatic tumor variants immunoselected by T lymphocytes originate from the coordinated downregulation of APM components

Previous reports from our group indicated that the MHC class I phenotype of metastatic lung colonies produced by a mouse fibrosarcoma tumor clone (B9) were, depending on the immune status of the host, MHC class I negative in immunocompetent mice and MHC class I positive in immunodeficient athymic nude/nude mice. Now we report the identification of the molecular alterations responsible for the changes of MHC class I molecules in both situations. Metastatic nodes were analyzed for the mRNA level of H‐2 class I and β2‐microglobulin genes, and several gene components of the major histocompatibility complex (MHC) class I antigen‐processing machinery (APM). These included the genes coding for the low‐molecular‐weight proteins LMP2, LMP7, LMP10, the transporter associated with antigen processing (TAP‐1, TAP‐2), and calnexin, calreticulin, tapasin, PA‐28‐α, PA‐28‐β, ERP‐59 and ER‐60. Analyses with RT‐PCR showed that TAP‐1, TAP2, LMP‐2, LMP7, LMP10, tapasin and calnexin mRNA specific for these genes was absent in metastases produced in immunocompetent mice. In contrast, similar techniques with mRNA preparations obtained from metastatic nodes from immunodeficient mice showed that the mRNA expression level of these genes was highly positive. Interestingly, the MHC class I‐positive or negative phenotypes of the metastatic colonies correlated with in vivo immunogenicity. H‐2 positive metastasis grew more slowly than the H‐2 negative ones when injected intrafootpat in syngeneic immunocompetent animals and were finally rejected. These results provide evidence of the role of T cells in immune surveillance against tumors and identify a mechanism targeted by antitumor T lymphocytes to generate MHC class I‐negative tumor escape variants.

Squalene protects against oxidative DNA damage in MCF10A human mammary epithelial cells but not in MCF7 and MDA-MB-231 human breast cancer cells

Until now, very little has been known about the antioxidant capacity of squalene and its effect on human breast tumourigenesis. In the present work, we investigated squalenes scavenging properties and its effect on cell proliferation, cell cycle profile, apoptosis, reactive oxygen species (ROS) level and oxidative DNA damage, using human breast cell lines. Our results showed that squalene neither possesses scavenging activity nor significantly alters cell proliferation rates, the cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A), minimally invasive (MDA-MB-231) breast cancer cells, and highly invasive (MCF7) breast cancer cells. However, we found that squalene did exert the following effects on MCF10A epithelial cells in a dose-dependent manner: (a) it decreased intracellular ROS level, (b) it prevented H(2)O(2)-induced oxidative injury, and (c) it protected against oxidative DNA damage. Interestingly, squalene did not exert these effects on MCF7 and MDA-MB-231 cancer cells. Therefore, our data suggest that squalene, found in high amounts in virgin olive oils, could be partially responsible for the lower incidence of breast cancer in populations that consume the Mediterranean diet due to its protective activity against oxidative DNA damage in normal mammary cells.

Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A) or breast cancer cells (MDA-MB-231 and MCF7). We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS) level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

Bioactive properties of the main triterpenes found in olives, virgin olive oil, and leaves of Olea europaea.

Oleanolic acid, maslinic acid, uvaol, and erythrodiol are the main triterpenes present in olives, olive tree leaves, and virgin olive oil. Their concentration in virgin olive oil depends on the quality of the olive oil and the variety of the olive tree. These triterpenes are described to present different properties, such as antitumoral activity, cardioprotective activity, anti-inflammatory activity, and antioxidant protection. Olive oil triterpenes are a natural source of antioxidants that could be useful compounds for the prevention of multiple diseases related to cell oxidative damage. However, special attention has to be paid to the concentrations used, because higher concentration may lead to cytotoxic or biphasic effects. This work explores all of the bioactive properties so far described for the main triterpenes present in virgin olive oil.

Antioxidant and antiatherogenic activities of pentacyclic triterpenic diols and acids

The present paper aimed to test the potential cardioprotective activity of four pentacyclic triterpenes, uvaol, erythrodiol, oleanolic acid and maslinic acid, widely distributed throughout the vegetable kingdom. For this purpose, their antioxidant and antithrombotic activities related to LDL particles have been in vitro evaluated. Results demonstrated that maslinic acid, uvaol and erythrodiol exert antiatherogenic effect while no effect was observed for oleanolic acid. Specifically, maslinic acid has shown the most potent dose-dependent antioxidant effect and did not have antithrombotic properties, whereas uvaol and erythrodiol exhibited both antioxidant and antithrombotic activities. In addition, antioxidant mechanisms of action were determined. While maslinic acid possesses dual activity acting as scavenger of free radicals and as copper chelator, uvaol is able to form a complex with copper and erythrodiol seems to behave as a retarder antioxidant. In conclusion, dietary triterpenes may exert a cardioprotective effect by different mechanisms of action related to antioxidant and antithrombotic activities.

Triterpenic content and chemometric analysis of virgin olive oils from forty olive cultivars.

Forty olive cultivars (Olea europaea, L.) from the World Olive Germoplasm Bank Collection of Cordoba (Spain) were studied for their oil triterpenic dialcohol (uvaol and erythrodiol) and acid (oleanolic, ursolic, maslinic) composition. Dialcohol content ranged from 8.15 to 85.05 mg/kg, erythrodiol being the most predominant (from 5.89 to 73.78 mg/kg), whereas uvaol content was found at lower levels (from 1.50 to 19.35 mg/kg). Triterpenic acid concentration oscillated between 8.90 to 112.36 mg/kg. Among them, ursolic acid was found at trace levels, while the mean values of oleanolic and maslinic acids ranged from 3.39 to 78.83 mg/kg and 3.93 to 49.81 mg/kg, respectively. The variability observed for both triterpenic dialcohols and acid content was emphasized by principal component and cluster analyses. Both analyses were able to discriminate between oil samples, especially by erythrodiol, oleanolic acid, and maslinic acids. Regarding these results, we conclude that the virgin olive oil triterpenic fraction can be considered as a useful tool to characterize monovarietal virgin olive oil.

Detection of circulating tumor cells in the context of treatment: prognostic value in breast cancer.

Circulating tumor cells (CTCs) in patients with breast cancer can be regarded as the pre-stadium of clinically manifest distant metastases. Here we present results on CTCs determination in peripheral blood (PB) of breast cancer patients in the context of treatment. Ninety-two patients were enrolled onto a prospective, unicenter study, and 71 of those are the focus of our analyses. CTC assessment was performed by isolating cytokeratin-positive (CK) cells by immunomagnetic techniques, with further identification by immunocytochemical methods. CTCs were detected in 47 (66%) patients: 35 with primary breast cancer, and 12 with metastatic disease. Five (14.3%) of those patients with primary cancer and CTCs showed first disease progression or died. Eleven (91.6%) of those patients with metastatic disease and CTCs before chemotherapy, died, During chemotherapy, >6 CTCs was correlated with a worse prognostic of disease in patients with metastatic disease (p=0.05). Four weeks after chemotherapy, 59 patients underwent a follow-up assessment. CTCs were detected in 54.2% of those patients. CTCs levels, and not the presence of CTCs alone, was associated with progression free of disease (p=0.052) and showed borderline significance with overall survival (p= 0.071). The differential prognostic and overall survival showed between patients with and without elevated CTCs before and at the end of chemotherapy, is of special interest in patients without clinical evidence of metastasis.