**7. Cancer**

Abundant studies offer evidence that oxidative stress, chronic inflammation, and cancer are closely linked. In response to harmful stimulation, such as pathogenic invasion, mechanical injury, and toxicity, the recruitment of inflammatory cells increases the release and accumulation of ROS at the site of damage [161]. This involves the activation of transcription factors, including NF-κB, signal transducer and activator of transcription 3 (STAT3), MAPK, and hypoxia-inducible factor 1 α (HIF1 α). These transcription factors coordinate the production of inflammatory mediators, including cytokines and chemokines, and COX2, which lead to the recruitment and activation of leukocytes and trigger the same key transcription factors in inflammatory cells, stromal cells, and tumor cells, resulting in more inflammatory mediators being produced and a cancer-related inflammatory microenvironment being generated and propagated [162].

The association between nutrition and oxidative stress may have an important role in cancer and cancer stem cell progression, as well as in therapy [163]. Over the last few years, many in vitro and in vivo studies have demonstrated that olive oil phenolic alcohols and their secoiridoid derivatives possess anticarcinogenic capacities (in many cases not mediated by molecular mechanisms directly related to their anti-oxidant activity) by blocking tumor angiogenesis [164], inhibiting proliferation and invasion [165–168], inducting apoptosis [169,170], and regulating inflammatory response [171], among others. The molecular mechanisms exerted in vitro and involved in these effects have been recently reviewed. While the exact underlying anticancer molecular mechanisms of OLE, OC, and HT are still not fully known, evidence continues to accumulate. For instance, OC had a notable cytotoxic activity in human melanoma cells but not in normal dermal fibroblasts, accompanied by a significant inhibition of ERK1/2 and AKT phosphorylation and downregulation of Bcl-2 expression [172]. In this sense, not only did OC induce cell growth inhibition more effectively than classical commercially available COX inhibitors, but it also inhibited colony formation and induced apoptosis (PARP cleavage, activation of caspases 3/7, and chromatin condensation) in HCC and CRC cells, whereas it was not toxic to primary normal human hepatocytes. In addition, OC treatment induced DNA damage, increased intracellular ROS production and caused mitochondrial depolarization, in a dose dependent-manner [173]. Finally, OC showed a potential beneficial effect in suppressing growth of hormone-dependent breast cancer and improving sensitivity to tamoxifen treatment [174]. As for OLE, treatment of HepG2 human hepatoma cells inhibited cell viability and induced apoptosis (upregulation of BAX and downregulation of Bcl-2), through activation of the caspase pathway and the modulation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway, suppressing the expression of activated AKT [175]. In addition, a combination (compared to separate exposures) of OLE and cisplatin showed enhanced antitumor activity against HepG2, resulting in further elevation of NO content and of the pro-nerve growth factor (NGF)/NGF balance, accompanied by an upregulation of caspase-3 and a downregulation of *MMP*-7 gene expressions, in a dose-dependent manner [176]. Regarding HT, this phenolic compound showed chemopreventive properties by preventing DNA damage in PBMCs and inhibiting (to different extents) proliferation of breast (MDA and MCF-7), prostate (LNCap and PC3), and colon (SW480 and HCT116) cancer cell lines [177]. Moreover, in papillary (TPC-1, FB-2) and follicular (WRO) thyroid cancer cell lines, high doses (with respect to other cancer cells lines) of HT reduced cancer cells viability by promoting apoptotic cell death via an intrinsic pathway [178]. HT and 2HT colonic metabolites (phenylacetic and hydroxyphenylpropionic acid) caused cell cycle arrest and promoted apoptosis in HT-29 and Caco-2 cells [179].

The modulation of the senescence-associated inflammatory phenotype has been suggested to be an important mechanism action of olive oil phenols. Cellular senescence, a process that restricts proliferation of damaged or premalignant cells, also plays a role in aging and age-related diseases, and represents an interesting therapeutic target [180]. In a recent study in pre-senescent human lung (MRC5) and neonatal human dermal (NHDF) fibroblasts, 4–6 weeks of treatment with 1 μM HT or 10 μM OLE aglycone (OLE) reduced β-galactosidase-positive cell number and p16 protein expression, IL-6, metalloprotease secretion, COX-2 and α-smooth-actin levels. In NHDF, OLE and HT treatment counteracted senescence-related rises in COX-2 expression, NF-κB protein level, and nuclear localization. In addition, pre-treatment with these phenolic compounds prevented TNFα-induced inflammatory effects in these cells [181].

Of note, studies of cancer development and dietary prevention are very difficult to carry out in humans, due to the paucity (or absence) of surrogate markers to be modulated by such interventions. Therefore, even though epidemiological, in vitro, and animal data do sugges<sup>t</sup> chemopreventive effects of olive oil phenolics, this hypothesis might never be confirmed in humans. Nevertheless, studies by Machowetz et al. [108] and Salvini et al. [107] in healthy males and postmenopausal women, respectively, have reported reduced oxidative DNA damage after short-term ingestion of phenol-rich olive oil. More recently, the PREDIMED trial reported a diminution in the incidence of breast cancer following long-term consumption of a phenol-rich olive oil-supplemented Mediterranean diet as compared to a low-fat control diet [22].

## **8. Rheumatic Diseases**

There are more than 200 different conditions that are labelled as rheumatic diseases, including rheumatic arthritis, systemic lupus erythematosus, and osteoarthritis (OA), among others. One of the major characteristics of rheumatic diseases is chronic inflammation and autoimmunity, which consequently leads to tissue destruction and reduces patient mobility [182]. Immune cells play a key role in inflammation due to involvement in initiation and maintenance of the chronic inflammatory stages. In particular, circulating monocytes that may differentiate towards macrophages or dendritic cells are able to produce proinflammatory cytokines and mediators (including ROS and COX-2), attracting T and B cells which contribute to maintaining the inflammatory process and eventually to tissue destruction.

Several in vitro and in vivo studies have been carried out with models of chronic inflammation and autoimmunity exposed to olive oil phenolics. LPS-exposed J774A.1 macrophages treated with olive oil biophenol extracts showed reduced iNOS and COX-2 expression (100 μg phenols/mL), and NO release in a dose-dependent manner (50–150 μg/mL) [183]. Furthermore, OC repressed MIP-1 α, IL-6, IL-1β, and TNFα levels, as well as GM-CSF protein synthesis and LPS-induced NO production in this cell line [184]. In a collagen-induced arthritis mice model, an EVOO biophenol extract significantly reduced the levels of proinflammatory cytokines, COX-2, and microsomal prostaglandin E synthase-1, inhibiting c-Jun N-terminal kinase, p38 and STAT-3, and reducing NF-κB translocation [185]. In the same mice model, intake of a HT acetate-supplemented diet significantly prevented arthritis development and decreased serum IgG1 and IgG2a, cartilage olimeric matrix protein (COMP) and metalloproteinase-3 (MMP-3) levels, as well as pro-inflammatory cytokine levels (TNFα, IFN-γ, IL-1β, IL-6, and IL-17A). The activation of JAK/STAT, MAPKs, and NF-κB pathways were drastically ameliorated, whereas Nrf2 and HO-1 protein expressions were significantly up-regulated [186]. In male Wistar rats with induced rheumatoid arthritis, supplementation with HT-enriched refined olive oil led to decreased histological damage, as well as reduced COX-2 and iNOS expression [187]. OA progression is characterized by increased NO production, which has been associated with cartilage degradation. OC and its derivatives decreased MIP-1 α and IL-6 levels [184], as well as lipopolysaccharide-induced NO synthase (NOS2) synthesis in ATDC-5 chondrocytes [188]. Although a consensus on the actual role of autophagy in OA has not been reached, several studies showed it is decreased in OA, and its activation is protective against OA [189]. HT increased markers of autophagy and protected human C-28/I2 and primary OA chondrocytes exposed to hydrogen peroxide from DNA damage and cell death induced by oxidative stress. This autophagy-inducing effect is engaged through SIRT1-dependent and -independent mechanisms [190]. In a pristane-induced systemic lupus erythematosus (SLE) mice model, administration of EVOO containing high levels of phenolic compounds (600 ppm) reduced renal damage and MMP-3 serum and PGE2 levels in the kidney, as well as proinflammatory cytokine production in splenocytes, while up-regulating Nrf-2 and HO-1 protein expression and the activation of JAK/STAT, MAPK, and NF-κB pathways [191]. Moreover, in PBMCs from patients with SLE and healthy donors, the phenolic fraction of EVOO modulated cytokine production (IFN-γ, TNFα, IL-6, IL-1β, and IL-10) and attenuated induced T-cell activation, possibly via NF-κB signaling pathway, as increased expression of I-kappa-Bα and decreased extracellular signal regulated kinase phosphorylation accompanied these anti-inflammatory and immunomodulatory regulations [192].

To date, very few human studies (to the best of our knowledge) have been performed to ascertain the potential pharma-nutritional activity of olive biophenols in rheumatic disorders. Conceivably, their anti-inflammatory properties should augmen<sup>t</sup> the habitual pharmacological therapy of such diseases and contribute to increase patient wellbeing. In this context, supplementation of a HT extract to early-stage knee OA subjects for 4 weeks improved the pain measurement index and the visual analog scale score [109].
