Hydroxytyrosol protects from aging process via AMPK and autophagy; a review of its effects on cancer, metabolic syndrome, osteoporosis, immune-mediated and neurodegenerative diseases

Depósito de Investigación de la Universidad de Sevilla https://idus.us.es/ This is an Accepted Manuscript of an article published by ELSEVIER in : PHARMACOLOGICAL RESEARCH, Vol. 123, on 2019, available at: https://doi.org/10.1016/j.phrs.2019.03.005” Copyright 2019 Elsevier. En idUS Licencia Creative Commons CC BY-NC-ND 1 Hydroxytyrosol protects from aging process via AMPK and autophagy; a review of its effects on cancer, metabolic syndrome, osteoporosis, immunemediated and neurodegenerative diseases Rocío M. de Pablos a,1 , Ana María Espinosa-Oliva a,1 , Ruth Hornedo-Ortega b , Mercedes Cano c , Sandro Arguelles c,⁎ a Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Seville, and Institute of Biomedicine of Seville (IBiS), Hospital Universitario Virgen del Rocío/CSIC, Seville, Spain b MIB, Unité de Recherche Enologie, EA4577, USC 1366 INRA, ISVV, Université de Bordeaux, Bordeaux, France c Department of Physiology, Faculty of Pharmacy, University of Seville, Seville, Spain ⁎ Corresponding author at: Department of Physiology, Faculty of Pharmacy, University of Seville, Prof. García González, 2. 41012 Seville, Spain. E-mail address: [email protected] (S. Arguelles). 1 Equal first authors. ABSTRACT Aging is a complex process. It is considered a risk factor for several diseases such as cancer, neurodegenerative diseases, cardiovascular diseases, and diabetes, most of which have an oxidative and inflammatory base. Given that life expectancy is increasing, there is a present interest in the search for anti-aging strategies that allow healthy aging. Interestingly, in Spain, where the Mediterranean Diet (MD) is the reference food pattern, life expectancy will have the highest average by 2040. This diet is characterized, among other items, by virgin olive oil intake, which contains between 50–200 mg/kg of hydroxytyrosol, a major polyphenolic component of olive oil. Hydroxytyrosol is formed by the hydrolysis of oleuropein during the maturing of olives, storage of olive oil, and preparation of table olives. It is a yield of oleuropein by microbiota action in the organism after virgin olive oil consumption. The daily intake in context of the MD is estimated to be around 0.15 and 30 mg/day. In the last few years, hydroxytyrosol has received increasing attention due to its multiple pharmacological activities, such as antioxidants, anti-inflammatory and pro-apoptotic activities. It has also been the focus of extensive research regarding its bioactivity. In this sense, hydroxytyrosol is under consideration for the development of new antiaging strategies. In this review we will summarize the potential anti-aging effects of hydroxytyrosol and its protective role in several agerelated diseases. Keywords: Aging, Senescence, Hydroxytyrosol, Mediterranean diet, AMPK, Sirtuin, Autophagy 2 1. Introduction The term aging is usually defined as the deterioration of various physiological systems (cognitive, endocrine, and motor, for example). Consequently, their functions lead to an increased vulnerability to environmental factors, a higher disease risk and eventually death [1,2]. Aging, of course, implies a passage of time and although it is a universal process, the degree of deterioration can vary significantly from one individual to another. This process is one of the most complex and devious of any biological phenomenon since it includes changes in mitochondrial functioning, autophagy, oxidative stress, altered energy metabolism, accumulation of altered proteins, impaired capacity of the organism to face challenges, etc. Many pathologies, such as diabetes, cancer, osteoporosis and neurodegenerative and cardiovascular diseases and some immunological changes are associated with aging [3–8]. These diseases are usually difficult to treat because their progression is slow and clinical signs do not appear until mid-life or old-age when cell loss is significant and irreversible. According to the World Health Organization (WHO) between 2015 and 2050, the percentage of people over 60 years old will go from 12 to 22% (2000 million). By 2020, the number of people over 60 will be higher than the number of children under 5. The progressive increase of age-related diseases associated with life expectancy increase will represent a challenge for world-wide public health systems. Thus, until more effective treatments and reliable diagnostic methods are found, prevention remains one of the best strategies to combat age-related diseases. If a drug or combination of drugs delays progression of these diseases or prevents them, the life span will be extended, along with a healthy more comfortable aging. Understanding the molecular pathways involved in the aging process is crucial in developing new anti-aging therapies. A healthy aging and a lower incidence of age-related diseases have been associated with the Mediterranean and Asian Diets in several studies. In this context, the aim of this review is to summarize the potential beneficial effects of hydroxytyrosol (HT), an important polyphenolic compound found in olive oil. We also discuss some nutritional aspects of HT, as well as its key role in modulating several cellular and molecular mechanisms of aging. 2. Mediterranean Diet and hydroxytyrosol The Mediterranean Diet (MD) is a cultural, historical, social, territorial and environmental patrimony that has been passed on from generation to generation for centuries and that is intimately linked to the lifestyles of the Mediterranean people. In fact, since 16 November 2010, the MD has been inscribed on the representative UNESCO list of the intangible cultural heritage of humanity (https://ich.unesco.org/). The MD was first defined by Ancel Keys based on the study of dietary patterns of Greek and Italian populations during 1960s. It was defined as a diet characterized by a low saturated fat intake and a high vegetable oil intake. However, over the years, this definition has been extended, emphasizing food and nutrients as key components, frequency of foods (daily, biweekly), abundance (high, moderate, low) and importantly the serving size [9]. Nowadays, the MD is more specifically defined by a high intake of extra virgin (cold pressed) olive oil (EVOO), vegetables and fruits, whole grain cereals, nuts and legumes. These along with a moderate intake of fish, dairy products and red wine, and with a low intake of eggs, red meat and sweets define the MD. Likewise, the energy and nutrient content of this Diet has been extensively quantified. For example, based on different data from multiple studies, it has been calculated that the MD provides an average of 2221.27 Kcal/day distributed in 37% energy from total fat (19% from monounsaturated fatty acids (MUFA), 5% from polyunsaturated fatty acids (PUFA) and 9% from saturated fatty acids (SFA), 15% from proteins and 43% energy from carbohydrates [10–12]. An essential component of MD is olive oil, which is rich in polyphenolic compounds; the total polyphenol intake has been estimated at 90.4 mg daily (11% of total intake) [13]. These polyphenolic compounds represent an enormous family (more than 8000 compounds described) of bioactive molecules, which are exclusively present in the plant kingdom. They are classified into two large groups: non-flavonoids and flavonoids, which are further subdivided into different subgroups (flavonols, anthocyanins, procyanidins (condensed tannins), flavononols and flavones, phenolic acids, stilbenes and hydrolysable tannins) in function of the number of phenolic rings that they possess as well as the location of the substituents [14]. These compounds are well known for the broad range of 3 biological properties that have been endorsed: antioxidant, anti-inflammatory, neuroprotective, cardioprotective and antiobesity, among others [15–20]. Olive oil composition is divided into two fractions: a saponificable fraction (98–99% of the total weight), formed mainly by triacylglycerides, oleic acid (18:1n-9) being the main component. The other fraction is the unsaponificable fraction (2% of total weight), with more than 230 chemical compounds, including polyphenols. The chemical composition of the phenolic fraction of olive oil includes both lipophilic (as tocopherols) and hydrophilic phenols [21]. HT, tyrosol and their secoiridoid derivatives (oleuropein, oleuropein aglycone and elenolic acid dialdehydes) are components of hydrophilic phenols [22,23]. Olive oil phenolic compounds can range between 100 and 600 mg/kg. These quantities are strongly determined by different factors such as olive variety and fruit maturity, age of the tree, olive oil processing, agricultures techniques as well as soil composition and the climate. Approximately, half of this amount corresponds to HT and its derivatives (oleuropein and tyrosol) [21,24]. The relationship between MD and disease prevention has been widely investigated, its cardioprotective action being the first and the most studied effect. Almost 40 years ago, the “Seven-Countries Study” noted that Crete farmers, despite the consumption of large amounts of fat, had the lowest cardiovascular mortality rate in the study [25], relating this association to the MD pattern. In addition, the “Lyon Diet Heart Study” explored the role of MD on myocardial infarction survivors, concluding that the MD may reduce the rate of recurrence up to 46 months. More recently, the “PREvención con DIeta MEDiterranea” (PREDIMED) study has demonstrated that a MD supplemented with EVOO or nuts (key components of the MD) diminishes the incidence of cardiovascular events in persons with high cardiovascular risk by approximately 30% [26,27]. Furthermore, other PREDIMED sub-studies have shown that the MD can modulate inflammatory markers or the expression of genes involved in low-density lipoprotein (LDL)-oxidation and can prevent the onset of other chronic diseases such as metabolic syndrome (MetS), diabetes and cancer [28–30]. Moreover, a great number of studies have associated MD with longer survival and the prevention of other age-related diseases in different populations [31–33]. Thus, the consumption of a typical MD has been associated with more than 50% lower rate of all causes of mortality and a significantly longer life expectancy [34,35]. More recently, it has been shown that the MD pattern also has a preventive role in neurodegenerative disorders and cognitive dysfunctions. Particularly this diet has been linked to a reduction in cognitive decline and a reduced risk of dementia, specifically Alzheimer (AD) and Parkinson disease (PD) [36–39]. These potential MD effects are closely related to the characteristically high intake of fruits and vegetables, red wine and olive oil, which are foods rich in polyphenolic compounds [40–43]. Based on the large numbers of clinical studies, the European Food Safety Authority (EFSA) authorised a claim about the benefits of the daily ingestion of olive oil polyphenols in November 2011 (www.efsa. europa.eu/). The EFSA Panel proposed the intake of 5 mg/day of HT and its derivatives to obtain these effects. These quantities can easily be reached within context of a MD and the consumption of moderate amounts of olive oil. As mentioned above, the major polyphenolic compound in EVOO is HT or 2-(3,4-dihydroxyphenyl) ethanol, a phenolic compound with a typical catechol structure and a molecular mass of 154.16 g/mol [44–46]. Significant amounts of HT can also appear in wine [47]. This presence in wine is attributed to yeasts and their capacity to produce HT from tyrosine during alcoholic fermentation [48]. Taking into account both sources, after consuming 50 mL/day of EVOO and 100–200 mL/day of wine, between 0.15 and 30 mg/day of HT can easily be achieved [49]. In addition, HT is a product of oleuropein hydrolysis, a reaction that occurs during the maturation and storage of olive oil, the preparation of table olives and under gastric conditions [50]. Besides, it has been demonstrated after in vitro and in vivo colonic fermentations that microbiota also has the ability to form HT from oleuropein [51]. Thereby, the exposure to HT results not only from its intake (EVOO, wine), but also from a significant degree of ingested oleuropein and its aglycone. Furthermore, HT is a known dopamine metabolite. In fact, after red wine ingestion, it has been demonstrated that the interaction between ethanol and dopamine leads to the formation of HT [52], suggesting that alcohol is an indirect promoter of endogenous HT production [53]. There is strong evidence that HT is bioavailable in a dose-dependent manner and with a maximum plasmatic concentration of 5–30 min after administration/ingestion of EVOO and VOO. This concentration has a plasma half-life of 1–2 min in both rats and humans [45,54–59]. After ingestion, more than 40% of the HT can be absorbed into the small intestine and colon [44,45,50]. Interestingly, HT absorption depends on the vehicle of 4 administration, or food matrix, in the sense of olive oil being the most effective when compared to an aqueous solution, this fact being related to the protection of antioxidants [55]. In spite of HT, absorption is reasonably good while its total bioavailability is poor (5–10%). This is due to the extensive first-pass metabolism in the gut and liver [60]. The enzymes of phase II reactions, also named conjugation reactions (uridine 5′-diphosphoglucuronosyl transferases and sulfotransferases) form glucuronide and sulfate metabolites that have been identified in plasma. Therefore, HT acetate can be formed by acyltransferase activity [61]. In addition, other plasmatic metabolites such as 3,4-dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenylacetic acid have also been detected in animals after oral or intravenous HT administration [45,54]. More recently, ortho-methyl products of HT (homovanillic alcohol and acid), glucuronide derivatives and glutathionyl conjugates have also been detected after oral consumption of VOO (25 mL) [62]. It is noteworthy that HT sulfate has been quantified as the main circulating metabolite in rat plasma, while both HT sulfate and HT acetate sulfate metabolites are predominant in human plasma [61,63]. In fact, HT-sulphate-3′ has been selected as the best biomarker of olive oil ingestion [64]. Additionally, HT is also highly metabolized by the gut microbiota, which transforms part of the absorbed HT into hydroxylated phenylacetic acids phenylacetic acid, 2- (4′-hydroxyphenyl) acetic acid and 2-(3′,4′- dihydroxyphenyl) acetic acid) (Mosele et al., 2014). Regarding HT excretion, up to 80% of the ingested amounts of this compound are recovered in urine and similarly about 90% are also conjugated [50,56,60]. Urinary HT metabolites are mainly glucuronidates and methylconjugates, with or without glucuronidation and free phenols. In humans, more than 50 metabolites have been identified after an intake of 50 mL of EVOO, where methylation and glucuronidation were the most common processes [59]. Moreover, HT and its metabolites have been identified in different tissues and organs such as muscle, testis, kidney, liver and even the brain. This fact shows that the molecule presents a wide distribution in specifically targeted organs and that it is able to cross the blood brain barrier [65]. 3. Effects of hydroxytyrosol on molecular mechanisms of aging Impairment of molecular mechanisms responsible for maintenance of cellular homeostasis that increases with age, will promote cell damage, increases non-senescent cell death and contributes to acceleration of both the aging process and chronic age-related pathologies. In fact, downregulation of signaling pathways, such as AMPK and SIRT1, responsible for preserving metabolic functions, stimulating the recycling process (autophagy or clearance of molecular damage) and decreasing cell waste, is one of the hallmarks of aging. Meanwhile, upregulation of these signaling pathways enhances longevity [1,2]. Other hallmarks of aging such as dysfunctional mitochondria, reduced proteostasis, genomic instability, decrease of stem cell regenerative capacity, enlarged persistent inflammation, generation of reactive oxygen species (ROS), cellular senescence, telomere erosion and chromatin and epigenetic alterations have all recently been summarized [66]. The authors discuss how regulation of these hallmarks of aging can improve longevity through modulating programmed cell death by apoptosis. A better understanding of cellular and molecular aging mechanisms, as well as their association with chronic age-related pathologies, is contributing to an innovative generation of anti-aging pharmacological therapy. For example, rapamycin, a natural bioactive compound isolated from Streptomyces hygroscopicus collected from Easter Island. Rapamycin was approved by the Food and Drug Administration (FDA) to avoid organ rejection after transplant operation and also has antiaging effects [67]. In fact, several laboratories have reported that ra pamycin increases the lifespan in several species and decreases different age-related dysfunctions through inhibition of the pro-aging mTOR pathway [67,68]. In this context, the use of natural bioactive compounds as a source of finding potential anti-aging molecules has in creased in the last 5 years. In this section we summarize recent evidence illustrating how HT can target one or more of the hallmarks of aging (Fig. 1). 3.1. Effects of HT on metabolic regulation Correct regulation of pathways responsible for energy metabolism is essential to the preservation of cell survival and to the improvement of longevity. In fact, the best-known anti-aging pathways, AMPK and SIRT1, are 5 upregulated in response to nutrient starvation, while proaging pathways, mTOR and IGF, are downregulated. 3.1.1. Effects of HT on AMPK AMPK has a fundamental role in modulating energy metabolism. Its dysregulation is associated with accelerated aging because of promoting inflammation, cancer and metabolic pathologies such as diabetes and obesity [69]. In this context, it has been reported that HT prevented type 2 diabetes brain damage through upregulation of AMPK [70]. The authors found that treatment with HT stimulated the activation of AMPK signaling, leading to increased neuroprotection in db/ db mice (which showed obesity and hyperglycaemia) and promoted neuronal survival in high glucose-induced neuroblastoma SH-SY5Y cell damage. It has also been reported that HT decreased lipid synthesis (e.g. fatty acid, cholesterol and triglycerides), for example, in hepatocytes, this effect being attributable to HT’s capability to upregulate AMPK signaling. AMPK decreases activity of acetyl-CoA carboxylase (ACC), 3-hydroxy-3-methyl-glutaryl-CoA reductase and diacylglycerol acyltransferase (HMGCR), thus avoiding hepatic lipid accumulation [71]. Similarly, in 3T3-L1 adipocytes, HT increased the activity of AMPK and genes involved in fatty acid oxidation such as Pparα, Cpt1 and Pparγ. Moreover, HT increased the phosphorylation-dependent inhibition of ACC and reduced levels of free fatty acids, which is responsible for promoting type 2 diabetes and stimulating insulin resistance [72]. Interestingly, despite AMPK activity not being examined, the effect of HT on lipid metabolism in glial cells has been studied, showing that HT decreased lipid synthesis by decreasing the activity of ACC and HMGCR [73]. The ability of HT to upregulate AMPK activity has also been reported in vascular endothelial cells. The authors found that HT-dependent upregulation of AMPK leads to an increased catalase (CAT) and transcription factor forkhead transcription factor 3a (FOXO3a) activity. This activity protected against H2O2-induced oxidative stress [74]. 3.1.2. Effects of HT on SIRT1 HT can also target additional signaling pathways involved in control of the aging process. For example, another well-known anti-aging molecule is SIRT1 [1,2]. Recently, it has been reported that in vascular adventitial fibroblasts stimulated with tumor necrosis factor-α (TNF-α), HT promoted the expression of SIRT1 leading to a stimulation of an anti-inflammatory response [75]. Mechanistically, the authors found that HT- mediated SIRT1 activation led to upregulated autophagy machinery by promoting the downregulation of the pro-aging AKT/mTOR pathway (the best-known inhibitor of autophagy). Using senescence-accelerated mouse-prone 8 (SAMP8) model, it was reported that high olive oil phenolic compound intake (including high concentration of HT) upregulated SIRT1. The upregulation led to stimulation of an antioxidant response and to a decrease of oxidative stress in the heart of SAMP8 mice [76]. At molecular levels, the antioxidant effect of SIRT1 has been linked to upregulation of nuclear factor-E2 and related factor 2 (Nrf2) [77]. In fact, although the intake of olive oil polyphenols in SAMP8 mice resulted in decreased lipid peroxidation and protein oxidation, Nfr2 and Nrf2-target genes were also increased, including glutathione-S-transferase (GST), γ-glutamyl cysteine synthetase (γ-GCS) and paraoxonase-2 (PON2). Bayram and colleagues suggested that HT is the polyphenol responsible for this protective effect because only HT increased Nfr2 and Nrf2-target gene such as PON1 in the cell culture [76]. Moreover, using other in vitro experimental models, HT promoted the upregulation of SIRT1. For example, in primary chondrocytes stimulated with H2O2, HT promoted chondrioprotection by increasing the nuclear localization of SIRT1 and inducing autophagy and the expression of autophagy substrate p62 [78]. Interestingly, D’Adamo and colleagues reported a potential mechanism responsible for HT-mediated SIRT1 activation. The authors found that SIRT1 was a direct target of miRNA-9. In fact, in chondrocytes, miRNA-9 repressed the expression of SIRT1 in response to H2O2 and HT. Moreover, depletion of miRNA-9 promoted SIRT1 expression and cell survival, while aberrant increase of miRNA-9 decreased SIRT1 expression and promoted cell death [79]. 3.1.3. Effects of HT on mTOR Although under physiological conditions mTOR is a pro-aging pathway and its inhibition increases the lifespan [67,68], mTOR also has important antiapoptotic effects, essential in preserving cell survival after injury. In fact, a transient increase of mTOR contributed to the promotion of cell survival after injury was induced by 6 aging-related ischemia/reperfusion (I/R) [80]. In this context, it has recently been reported that HT promoted cell survival against myocardial I/R injury by upregulation of the PI3K/AKT pathway (best-known activator of mTOR) [81]. However, the aberrant activation of the PI3K/PTEN/AKT/ mTOR pathway is a hallmark of aging and age-related diseases [82]. Indeed, in several cancer cells, HT induced downregulation of PI3K/ AKT, thus promoting G2/M cell cycle arrest and apoptosis in vitro, as well as decreased tumor growth and angiogenesis in vivo [83]. Interestingly, in some cancer cells (e.g. human colon cancer cells) HT promoted the upregulation of the PI3K/AKT pathway and decreased FOXO3a target genes, thus stimulating mitochondrial dysfunction and oxidative stress-induced apoptosis [84]. 3.2. Effects of HT on oxidative stress Upregulation of oxidative stress is associated with mitochondrial dysfunction, inhibition of autophagy and accelerated aging [1,2]. The ability of HT to decrease oxidative stress is one of its most reported properties (further reviewed in [85]). Indeed, HT treatment increased activity of FOXO3a, Nrf2 and Nrf2-target genes such as γglutamyl cysteine ligase (GCL), NAD(P)H quinone oxidoreductase (NQO1), heme oxygenase-1 (HO-1), glutation reductase (GR), glutation peroxidase (GPx) and CAT, all of which are essential to oxidative stress defense [85]. Moreover, recently, it has been reported that regular intake of HT in healthy volunteers improved the antioxidant defense (e.g. total antioxidant status, thiol group and superoxide dismutase (SOD)) and decreased nitrate and malondialdehyde [86]. Moreover, the increase of oxidative stress produced in high-fat diets (HFD)-fed mice in white adipose tissue was reduced by HT. The authors found that HT decreased the oxidation of lipids and proteins, while increased antioxidant defence such as reduced glutathione (GSH) content, GSH/GSSG ratio, Nrf2, GCL, GST, CAT, SOD, GPx) and GR. This protective effect of HT contributed to effective control of inflammation and lipogenesis [87]. In keratinocytes and fibroblasts, blue-light exposure promoted aberrant increase of ROS, which lead to DNA damage and premature aging. Recent reports show that HT prevented oxidative stress, improved DNA damage repair mechanisms and avoided the degradation of extracellular matrix components in keratinocytes and fibroblasts [88]. 3.3. Effects of HT on mitochondria dysfunction Mitochondrial dysfunction contributes to acceleration of the aging process because it increases the production of mitochondrial ROS (mtROS) resulting in aberrant accumulation of oxidative stress and oxidative damage. Thus, promoting age-related pathological states [1,2]. In different experimental models, it has been reported that HT attenuated mitochondrial dysfunction. For example, an uncontrolled increase of inflammation stimulates mitochondrial damage. In this sense, HT improved mitochondrial function in human umbilical vein endothelial cells, induced angiogenesis, and increased mitochondrial biogenesis, along with ATP production, mitochondrial membrane potential and ATP5β expression. Another report showed that HT also promoted the decrease of inflammatory cytokines, endothelial adhesion molecules, mtROS production, SOD activity and oxidative biomolecules damage [89]. We can find another example in the brain, where mitochondrial dysfunction is associated with age-related neurodegeneration. In fact, in rat brain, HT re-established Arsenic-induced mitochondrial dysfunction by increasing mitochondrial complex (I, II, IV) activities and upregulating antioxidant enzymes such as mitochondrial SOD (MnSOD) and CAT [90]. Interestingly, HT induces extension of chronological lifespan in human skin fibroblasts by increasing MnSOD and decreasing age-associated increase in mtROS levels [91]. Moreover, in the cardiovascular system, mitochondrial impairment is linked to age-related cardiac dysfunction. In cardiomyocytes, HT protected mitochondria against doxorubicin-associated mitochondrial dysfunction by avoiding mitochondrial disturbance, protecting mitochondrial electron transport chain complexes I-IV, improving the integrity of complex-III and decreasing oxidative stress and apoptosis-inducing factors [92]. Finally, Zheng and colleagues have shown that prenatal stress-induced mitochondrial dysfunction was diminished by HT, which induced upregulation of complex I–V components, avoided the decrease of Nrf2 expression and Nrf2-target gene, increased SOD2 and FOXO1/3, as well as decreased protein oxidation [93]. 7 3.4. Effects of HT on autophagy With regards to autophagy, a well-known biological process that contributes to longevity and decreases age-related pathologies [1,2], in human THP-1 monocytes, HT treatment increased autophagy markers (LC3II and p62), intensified the inhibition of mTOR/AKT pathways and promoted the activation of AMPK. Moreover, in response to lipopolysaccharide (LPS)-induced inflammation, HT blocked the release of proinflammatory molecules such as interleukin (IL)-6, IL-1β, TNF-α, HMGB1 and granzyme B [94]. Similarly, HT stimulated the induction of autophagy through upregulation of SIRT1 in LPS-induced acute lung injury in mice. The authors also found that the anti-inflammatory effect of HT was mediated by depletion of proinflammatory molecules such as TNF-α, IL-1β, IL-6, IL-10, and monocyte chemoattractant protein 1 (MCP- 1) [95]. HT also promoted autophagy markers (LC3 and Beclin) and the autophagic flux by inducing SIRT1 expression and inhibiting AKT/mTOR in vascular adventitial fibroblasts stimulated with TNF-α [75]. Interestingly, in most cases HT promotes autophagy by upregulating SIRT1, however a recent report showed that this HT-mediated autophagy stimulation is independent of SIRT1 because HT still induces autophagy in SIRT1-depleted cells. The authors suggested that the upregulation of p62 was responsible for increasing autophagic flux in those cells [78]. 3.5. Effects of HT on DNA damage/repair DNA repair machinery is essential for correcting self-damage to the DNA, and the maintenance of genomic stability and promoting longevity [1,2]. One of the most used assays for evaluating DNA strand breaks in eukaryotic cells is the comet assay. This essay showed that HT considerably diminished oxidative stress in noncancer cells (e.g. MCF10A) and avoided H2O2-induced DNA damage in both non-cancer (e.g. MCF10A) and breast cancer cellular types (e.g. MCF7, MDA-MB- 231) [96]. Similarly, the comet assay has been used to report that HT prevented DNA damage in human peripheral blood mononuclear cells and promyelocytic leukemia cells (HL60), [97]. Interestingly, in several cell types and in response to different DNA-damage inductors, HT avoided DNA damage. For example, HT-lipophilic analogues show significant protection against oxidative stressinduced DNA damage in whole blood cells evaluated using alkaline atypical comet assay [98]. HT also protected against acrylamide-induced cytotoxicity and DNA strand breakages in HepG2 cells [99]. Besides, HT stimulated the inhibition of UVB-induced DNA strand breaks in HaCaT cells [100]. It is possible that the wellknown antioxidant activity of HT could be one of the potential mechanisms responsible for avoiding DNA damage. However, we cannot discard additional potential mechanisms to explain the DNA damage repair capacity of HT. For example, more detailed studies using HT and analyzing specific markers of DNA repair machinery such as mismatch repair, base excision repair, single-strand break repair, nucleotide excision repair, homologous recombination or non-homologous end-joining are needed. 3.6. Effects of HT on epigenetic regulation The molecular mechanisms of epigenetic alterations and their contribution to the aging process have been reported by numerous groups and have been excellently summarized [1,2]. In this context, a few reports have shown HT-induced epigenetic modification affecting DNA methylation. For example, the endocannabinoid system, which has been linked to control of oxidative stress, inflammation, as well as regulation of the immune system [101], can be regulated by HT through epigenetic mechanisms in context of cancer. In human Caco-2 colon cancer cells, treatment with HT increased the expression of cannabinoid receptor 1 (CB1) by decreasing the methylation levels of cannabinoid type 1 receptor gene (CNR1). Decrease of CNR1 methylation by HT led to inhibiting proliferation in colon cancer cells. Since HT promotes upregulation of miRNA associated with colorectal cancer such as miRNA-23a and miRNA-301a, the authors suggested that these miRNAs contribute to upregulating CB1 [102]. Given that downregulation of CB1 has also been associated with age-related memory decline [103] and senescence [104], it could be possible that HT contributes to improved healthy aging and longevity. 8 4. Effects of hydroxytirosol on aging-related diseases Once molecular mechanisms are explained, through which HT contributes to the reduction of different aspects related to aging, in this section we summarize experimental results that suggest a potential use of HT against some age-related diseases (Tables 1 and 2). Moreover, interventional and observational trials showing the protective role of HT in several age-related diseases are indicated in Table 3. 4.1. Cancer Cancer is a convoluted chronic-degenerative disease characterized by processes in which normal cells are transformed into malignant cells by the acquisition of several properties such as reduced apoptosis and abnormal proliferation. Age is one of the higher risk factors for cancer development, which can be inhibited by the modulation of longevity pathways [105]. In Western countries, cancer is responsible for major morbidity and mortality, although its mortality has decreased during the last 15–20 years. Still, even with that decrease, more than 8.2 million cancer deaths were estimated for 2012 [106]. It has also been estimated that, by 2030, the number of new cases will increase to about 23.6 million. Many types of cancer are still incurable either because they are diagnosed late or because a definitive therapy is unknown. Besides, most therapies lead to serious side effects and toxicity because the drugs cannot distinguish between cancerous and normal cells. It is therefore necessary to find new drugs/compounds that cure or at least reduce the progression of the disease without affecting normal cells. In general, it has been estimated that many types of cancers might be avoided or cancer survivors increase simply by changing diet [107]. Therefore, an important goal for current nutritional research is identification and characterization of foods and their constituents that can prevent cancer [108–112], or stimulating cancer cell death, for example, by promoting anti-growth signaling or disrupting replicative immortality [113,114]. In this sense, olive oil phenols, including HT, have demonstrated a positive action on cell growth and proliferation, invasion, angiogenesis and migration (Fig. 2), showing an important role in the reduction of certain types of human cancers such as colorectal, thyroid, skin, digestive, breast, lung, blood, brain and cervical [115]. The focus on this compound intensified when a lower cancer incidence was observed in Mediterranean regions [116]. HT is known to have several anti-tumoral effects, including suppression of cyclooxygenase-2 (COX- 2), FAS and Bcl-2, induction of apoptosis, cell cycle arrest, reduction of ROS and pro-inflammatory cytokines production, plus reduced DNA damage (for details about molecular mechanisms, see reviews from [115,117,118]). These and other effects have been supported by both in vitro and in vivo studies. For example, in a recent paper Cruz-Lozano and colleagues showed that HT inhibited breast cancer stem cells and tumor cell migration by targeting epithelial-to-mesenchymal transition as well as transforming growth factor-β and Wnt/β-catenin signaling pathways in triple-negative breast cancer. This is the most aggressive breast cancer type, that with which treatments have less effect, resulting in high mortality rates. This finding has sparked new hope for fighting this type of cancer [119]. Moreover, breast cancer patients that combine adjuvant hormonal therapy with HT, omega-3 fatty acids and curcumin, have pain and inflammation alleviated in the early stages [120]. The potential anti-cancer effect of HT has also been found in other cancers such as pancreatic [121] and skin cancers [122]. The current pancreas cancer treatment increases survival only up to six months. In most cases, it damages normal tissues, unfavorably affecting the life quality of patients. Toxicity towards some pancreatic cancer cells (by apoptosis induction) from HT has been seen, so HT could be used in pancreatic cancer prevention [121]. In the case of skin cancer, HT has been presented as an alternative to topical chemotherapy through the development of HT-containing alginate bilayer films [122]. All these data make HT an excellent candidate as coadjutant therapy strategy in many types of cancer due to its anti-tumoral properties and low toxicity. 4.2. 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